Lupine Publishers | Raya Indigenous Livestock Husbandry Practices in the Highlands of Southern Tigray, Ethiopia

 Lupine Publishers | scholarly Journal of Food and Nutrition

Abstract

Raya indigenous livestock husbandry practices was conducted in Emba-Alaje Enda-Mekoni and Ofla Wereda of Southern Tigray, featured by mountain chains and located at 12°47’ N latitude 39°32’ E longitude. with the aim to determine constraints and opportunities that exist within the farming systems, for better targeted improvement and to design policies strategies to support peculiar livestock husbandry practice, since it is experiences of the greatest successes stories of developing country agriculture and one of the most unsung, especially in the disadvantaged marginalized areas. Single household respondent was used as sampling unit, using Proportional Probability to Size approach. Out of 156HHs, 73.5% were male headed while 26.5% female-headed. Educational status of HHs members was diverse that was composed of 12.8% educated while 41%HHs members were illiterate. Average family size was 4.6±1.84. 83.33%HHs used own family labour, while others use hired labour. Feeding, watering, barn cleaning, animal keeping, monitoring animal health, cow milking, and selling dung cake tasks of wives and children, while feed purchase, buying and selling animals were husband’s duty. Age at first calving was 3.5 years for local while 2.5years for exotic breeds and calving interval was similar 1.5 year. The average milk yield was 2±1 litres for Arado, 5±1 litres for jersey and 10±2 litres for Holstein Frisians. The average cattle herd size were 3+1 in urban, 4.67+4.93 in periurban and 3.75±2.12 in rural farms. There was significant (P< 0.05) difference for cattle breed in lactation length and milk yield but no remarked (p>0.05) difference in Wereda level. Housing system of the study areas were featured backyard compound in 62.18% of the respondents, partial shelter in 17.95% of the respondents and improved barn in 19.87% of the dairy farmer respondents. Alternative interventions for betterment of the indigenous husbandry practice is with the climate change are timely scenario.

Keywords: Raya, Indigenous livestock husbandry, Arado, Holestain fresian

Introduction

Domestication of ruminant animals and their use to produce milk, meat, wool, and hides represents one of the cornerstone achievements in the history of agriculture. The essential feature of the ruminant animal that has fostered its utility as a dairy animal is the presence of a large pre-gastric chamber where microbial digestion of feed particularly fibrous feeds not directly digestible by human, provides various fermentation products that serve as precursors for efficient and voluminous synthesis of milk. Without this symbiosis between animal and microbe, the dairy industry would not have developed, and indeed human culture would be vastly different in its food-gathering methods (Weimer and James, 2001).

In Ethiopia, the livestock production system, which is dominated by indigenous breeds of low genetic potential for milk production, accounts for about 98% of the country’s total annual milk production. The low productivity of the country’s livestock production system in general and the traditional sector in particular is mainly attributed to shortage of crossbred dairy cows; lack of capital by dairy producers, inadequate animal feed resources both in terms of quality and quantity; unimproved animal husbandry system; inefficient and inadequate milk processing materials and methods; low milk production and supply to milk processing centers; and poor marketing system. Making improvement interventions to the traditional sector is, therefore, crucial if development of the livestock sector of the country is targeted. Its large livestock population; the favorable climate for improved, high-yielding animal breeds; and the relatively diseasefree environment for livestock make Ethiopia to hold a substantial potential for dairy development. Considering the substantial potential for smallholder income and employment generation from high-value livestock products, development of the dairy sector can contribute significantly to poverty alleviation and nutrition in the country. With the present trend characterized by transition towards market-oriented economy, the dairy sector appears to be moving towards a takeoff stage [1].

Dairy enterprises are the “white gold” of many developing countries, creating pathways out of poverty while boosting better human nutrition and health, regular income generation, employment, crop farming, and natural resource management. The context for smallholder dairy development in Ethiopia has been changing rapidly, creating both new opportunities and challenges [2]. According to Mburu [3] characterization of smallholder dairy production systems in highlands is critical in understanding the constraints and opportunities that exist within the farming systems. It allows better targeting of dairy improvement research and development. Therefore, information obtained can be valuable for detailed analysis of constraints and opportunities found in smallholder dairy systems and to design policies and strategies to support smallholder dairy development programs in variable intensification that one has to be aware of the challenges of dairy which, is one of the greatest successes stories of developing country agriculture and one of the most unsung, especially in the disadvantaged marginalized areas.

The bulk of Ethiopian livestock’s provision to the economy is not properly identified in conventional national accounts as coming from livestock. These distortions are particularly acute for highland livestock production systems in which animal energy for transport and dung for fuel are as important as conventional milk and meat production [4] that confirmed less attention was given to the sector despite its indispensible contribution to the economy of the majority of dairy farmers and the nation.

Livestock production in Ethiopia is constrained by a multitude of technical, financial, institutional and socio-economic factors [5]. Coordinating inputs (knowledge, finance, social and political capital) of various actors and their expectations in a way to create best practices and innovations could contribute better exploitation of the resource [2]. ‘When there is no bridge, there is always other means!’ [1]. The marginalized disadvantaged dairy farmers did not have exposure and access to affordable improved technological facilities that enable livestock production ease and profitable; consequently they do act according to their local resources and custom which demand due focus and research.

In Ethiopia, particularly in the highlands of Southern Tigray where previous research is very meagre [5], the indigenous livestock husbandry system is very peculiar than any other areas since long period of time but the doubt is their extent of production in comparison to their demand, nutritional needs and economic values, that is why the objective of this paper has targeted on the main indigenous livestock husbandry practices in relation to the livestock resource potential. Thus this work was initiated with the following objectives:-

1. To identify indigenous livestock husbandry practices & constraints in the study area, and

2. To determine the livestock breed composition of the area

Materials and Methods

The research was conducted in Emba-Alaje Enda-Mekoni and Ofla Wereda of Southern Tigray, from December 01, 2011 to February 30 2012, which are featured by mountain chains, where Maichew of Enda-Mekoni is located at 12°47’ N latitude 39°32’ E longitude and an altitude of 2450m.a.s.l. It has a rainfall ranging from 600-800mm , temperature ranging 12-24oC, and relative humidity of 80% , which is highly variable from year to year and erratic in nature. The district is located on about 90-180km south of Mekelle city and 600-690Km north of the capital city Addis Ababa. The study area is also categorized as one of the populated highland areas of the country where land per household is 0.8h. Korem of Ofla lay on 12029’N latitude, 39o32’E longitude and that of Adishehu of Emba-Alaje is located on 120 56’N latitude and 39029’E longitude [6].

Single household respondent was used as sampling unit and sample size determination was applied according to the formula recommended by Arsham [7] for survey studies:

SE = (Confidence Interval)/(Confidence level) = 0.10/2.58 = 0.04, n= 0.25/SE² = 0.25 / (0.04)²= 156

Where, confidence interval=10% and confidence level=99%

Where: N- is number of sample size

SE= Standard error, that SE is at a maximum when p= q = 0.5, with the assumption of 4% standard error and 99% confidence level.

The total sample size was determined to be 156 for the household level interview. Proportional Probability to Size (PPS) approach for uniformity matters as Desalegn [8]. Three approaches namely, participatory rural appraisal for base line information and formal (diagnostic) survey using well-structured questionnaire, farm visit & group discussions of the entire system were used to generate qualitative & quantitative data.

A translated pretested semi-structured questionnaire was used to collect information on quantitative and qualitative data: Demographic situations, level of education, type of dairy breed, production performance, production objectives, variety of products, husbandry system, major production constraints, livestock disease incidences; opportunities for improvement and other related issues.. For the field survey, the method of data collection used was single- visit-multiple-subject survey. Data collected were analyzed using Microsoft Excel [9] and Statistical Package for Social Sciences [10] computer software program. Survey results were summarized using descriptive statistics like mean, standard deviation, and percents; mean differences were tested using student’s t.

Result

The results obtained on household characteristics are presented in (Table 1). As shown, 25.6% of the respondents were less than 40 years of age, 51.3% of them aged 41-60 years while those with the age of more than 60 were only 23.1%. Of the total households interviewed, 73.5% were headed by males the rest being female-headed. When the issue comes to literacy level, the educational status of the household members was diverse that was composed of 12.8% educated while 41% of the household members were illiterate (i.e., do not read and write). Average family size was 4.6±1.84 that ranged from 1 to 14. Labour use, 83.33% of the interviewed households used own family labour, where as the other proportion of them use hired labour in addition for dairy farming. Feeding, watering, barn cleaning, animal keeping, monitoring animal health, cow milking, and selling dung cake were performed mainly by wives and children, while feed purchase, buying and selling animals were responsibilities of the husband.

Milking Procedure Practiced

Milking twice per day (morning and evening) was the tradition followed by all households. Among the respondent dairy farmers, 25% of both urban and periurban dairy farmers practice zero grazing and milk their animals at a regular time of the day to supply the product according to their customers demand (Table 2). Whereas the rest of the proportion do not follow regular time of milking apart from maintaining the frequency. The housing systems, the cleaning processes and the procedures followed by the household are predominantly traditional. Udder washing was practiced by 10.89% respondents, of which 23.10% were from Emba-Alaje, 35.3% from Enda-Mekoni, 29.4% were from Ofla urban and 8.6% were Ofla rural areas who introduced cross breed cows.

Feeding Practice

Crop residues from teff, pulses, barley, wheat and maize and sorghum plus hay and natural pasture are the major feed resources the study area. Coping mechanisms practiced in the study areas during feed scarcity were moving to areas with available feed termed as ‘urna’, providing grass harvested from sloppy hills. The other important feed resources include spineless and thorny Cactus while some do practice forage development minimally. The crop residue conservation practices followed by the farmers are subject to nutritional losses. In the urban dairy farming, use of concentrate feeds is a potential alternative through which productivity of cows can be improved; however, the high cost was a limiting factor. Majority of the dairy farmers use leftover house hold feeds such as hull of grain after milling. Hatela (slurry from local brew) was another form of concentrate feed available (Figures 1 & 2).

Housing Systems

Housing system of the study areas were featured backyard compound in 62.18% of the respondents, partial shelter in 17.95% of the respondents and improved barn in 19.87% of the dairy farmer respondents. In Urban Emba-Alaje, 76.92% of the respondents practiced improved housing but not hygienic for they do not clean the barn because they deemed crucial bedding to absorb heat for the animals (Figure 3).

Calf Rearing

Cattle are kept in barns under normal circumstances and calves are kept in houses until they are strong enough to bear the extreme climatic phenomena. Young animals are managed in a traditional way. Suckling calves are kept separate from their dams, except when calves are used to stimulate milk letdown. Traditionally, calve suckling practice is believed to stimulate milk letdown, prevent teat blockage and softened the teat for ease of hand milking. If the calf dies, the hide is stuffed with cereal straw or grass with four legs made of sticks, rubbed by salt so that the dam would lick it to simulate the presence of the calf and stimulate milk letdown. Young children and females in general do mostly attend calves near encampments. Herders are well aware of colostrum feeding for the new born animals and understand the beneficial effect on health of the young.

In all the rural and periurban areas calves are herded in group by child and/or widowed of misery part of the community and encouraged by providing milk of every Wednesday termed as ‘tseba rebue’, while urban areas do practice tethering in backyards. Overnight, calves do spent in calf pen (urban and periurban) or in the normal household home (rural areas) isolated from their dams or herd. In local cows majority of the dairy farmers responded until the cow become dry of that rejects her calf from suckling was related with end of lactation period. But those owners of exotic do practice 4-6 months suckling before weaning. Traditionally, the herders use different types of weaning methods. Weaning is performed by piercing the nose of the calf with thorns, twisting up the nose skin of the calves to prevent suckling (as this causes pain when the wounded nose touches the teat) and smearing of teats with animal dung (Figures 4 & 5).

 

Record keeping

The most important record kept in the dairy farms was birth date that was considered in 44.9% respondents followed by 29.49% respondents to record amount of milk sold, 27.56% respondents used breeding record and 27.56% respondents used feed expenditure record, while 53.20% of the respondents do follow random husbandry practice. Breeding record, birth date and feed cost are recorded. Wereda level 72% of respondents from Enda-Mekoni, 33.33% respondents from Emba-Alaje and 32.76% of respondents from Ofla had record keeping trials (Table 3).

Milk Products Marketing

It was noticed that milk marketing was limited to urban and periurban areas but not in the rural districts. The major milk marketing challenges the respondents complained were 52.56% claimed cultural taboos and distance from market areas while 26.92% of the respondent dairy farmers blamed the discouraging market due to lower understanding of consumers to milk nutrition, poor talents of entrepreneurship of milk producers, and lack of road to transport milk from remote areas. Majority of the studied households reported that the demand for the milk products was high during dry season and low during wet season, besides to the fasting periods.

In the study area, the smallholders rear livestock for draught power, milk production, beef production and generate income through live animal sale, especially as a guarantee in case of risk. Also respondents indicated that cattle were used as manure for fertilizing the homestead farmland and compaction of seedbeds. Hide and skin of the animal was used either as source of cash income or used as household furniture such as grain storage, mat and to carry a baby on back of mothers locally termed as “delobo” Others: include manure, dung to smear floors and walls and also for fuel (for cooking purpose or to fire alternative thorny cactus feed). Concerning to dung utilization, 5% of Ofla Wereda respondents do practice biogas, while the rural Enda-Mekoni in vicinity to Ofla have exposure and were in infant stage unlike to Emba-Alaje Wereda where there was no dream of biogas. The interesting thing is dairy farmers exchange dung cake for hatela concentrate feed contracts in majority of urban dairy farms or else cover some part of household earning by selling particularly females of the household (Figure 6).

Age at first calving was 3.5 years for local while 2.5years for exotic breeds and calving interval was similar 1.5 year. The average milk yield was 2±1 litres for Arado, 5±1 litres for jersey and10±2 litres for Holstein Frisians. The average cattle herd size were 3+1 in urban, 4.67+4.93 in periurban and 3.75±2.12 in rural farms. The population of Holstein Frisian decreased from urban to rural while that of the Arado breed increased, indicating that dairy farming in rural destined on Arado while urban destined on Holstein Frisian breeds. Milking cows of the study areas were 23.1% out of 845 cattle owned by the respondents, which were composed of 631 local including Arado, Raya and Begait breeds and 214 crossbred of Holstein Frisian and Jersey upgraded cattle (Figure 7) (Table 4).

Animal Health Challenges

There was outbreak of FMD regional level, in particular, Emba- Alaje area but controlled due to regional vaccination campaign. In steep gorges of mountain area and less infrastructure, efficiency of the veterinary services or the veterinary personnel highly depends on the availability of facilities such as transportation, veterinary equipment, drugs. Besides, the farmers practice folklore medicine, to save their animals by bleeding, branding and use of herbal medicines. Urban dairy farmers do have better access to veterinary service that could be affordable in comparative to their income from milk. The steep gorges of the study area are part of animal and human hazard losses that enforced some farmers to stick on zero grazing. The author has also experienced to see severely broken or death of animals through falling in the steep gorges (Figure 8) (Table 5).

CBPP= Contagious Bovine Pleuro Pneumonia

Discussion

The mean value of family size in the study areas 4.6±1.84 persons was comparable to CSA [6] report which was 4.5 for Enda- Mekoni, 4.29 for Ofla and 4.36 persons to a household for Emba- Alaje. This slight difference might be the reflection of the steady growth of the population. The proportion of the households who participated in the dairy technology package was 28.8%. In terms of labour use, 83.33% of the interviewed households used own family labour, where as the other proportion of them use hired labour in addition for dairy farming. Feeding, watering, barn cleaning, animal keeping, regulating animal health, cow milking, churning milk, milk selling and selling dung cake were more of performed by wife and children, while feed purchase, buying and selling animals as well as medication activities (bleeding and branding), were responsibility of the husband. Sell and purchase of dairy animals belong to the spouses more of men while women discharge feeding, milking and dairy products processing and selling. Herding to adolescents or hired in free grazing on communal natural pastures that constituted almost the only feed resource for all rural dairy farmers. Similar work by Girma, et al. [11] characterized that children are the primary care takers of cattle at day time. Rural dairy farms are characterized by roofless fenced enclosures to keep cattle during night times; calves being separated from adults and housed in the same shelter with households, however, dairy farming packaged households do abide by zero grazing and modified shelter for the hybrid Holstein Frisian cows.

Milking cows of the study areas were 23.1% out of 845 cattle owned by the respondents, which were composed of 631 local including Arado, Raya and Begait breeds and 214 crossbred of Holstein Frisian and upgraded Jersey cattle The result is indifferent from MoA (2004) report in Ethiopia that 11.82% of 2990 cattle population in 1998 was milking cows. That could be due to time difference and business mindedness of dairy farmers in urban agriculture now than draught oxen focus by the then time. The population of Holstein Frisian decreased from urban to rural while that of the Arado breed increased, indicating that dairy farming in rural destined on Arado while urban destined on Holstein Frisian breeds. Milking twice a day is similar to the milking frequency practiced in many parts of the country. Time of milking is normally early morning and late evening that is consistent with Sintayehu (2008). But time of the day particularly morning hours could vary that milking is delayed during cool seasons.

Average age at first calving was 3.5 years for local, while 2.5 years for exotic breeds and calving interval was similar 1.5 year. The lactation length was averaged 6±1 months for local cows while 8±1 months for exotic breeds that matched with Dawit (2009) report in Eastern Tigray who also summarized, milk yield of local breeds from 1.8±0.4 in Arado to 5±0.5 of Begait breeds. The average milk yield was 2±1 litres for local breeds, 5±1.5 litres for hybrid jersey and10±2 litres for hybrid Holstein Frisians. There was significant (P< 0.05) difference for cattle breed in lactation length and milk yield but no remarked (p>0.05) difference in Wereda level. Highest lactation length recorded in Maichew Holstein Frisian was 2 years, contrary to the universal record of 10 months exotic breeds, actually the cows displayed no observed heat. The study result disagreed with Mulugeta [12] who reported average daily milk off take from local cows 1.09 litres and crossbred cow 5.97 litres, with overall lactation length of both local and crossbred cows was 7.52±1.64 months as per farmer’s statements. Adebabay [13] recorded local cow’s milk yield of 1.46kg/cow/day. Genzebu (2012) in northern Tigray also added that Arado cows give an average milk yield of 1 - 2 liters/day for an average lactation period of 7.3 months.

In close affinity to Asfaw (2010) work in Arsi zone, generally more number of services per conception was reported using AI as compared to natural mating, attributed to inefficient AI services that included poor quality semen, poor heat detection techniques and inaccurate AI services. The same is true in feeding system that dairy producers practiced inadequate crop residue storage that hinders productivity of the animals. Similar to the reports of FAO, IDF [14] and Thapa (2000) dairy production was influenced by feed problem, poor animal health services and shortage of drugs, dissemination of poor genetic material, poor government attention to dairying, unreliable AI service, working land shortage to expand and/or forage development, market problems for dairy products, financial problem (absence of credit), waste disposal, lack of recording system (poor information flow), lack/poor extension service & training, lower understanding of the respondent, poor hospitality of AI/ veterinary renders. Traditional medication practice such as bleeding and hot branding that damage hide economy of the nation for unreliable remedy could be minimized as remarked PPLPI [15] by pen side diagnostics for common diseases.

Conclusion and Recommendations

Livestock production plays an important role in the socioeconomic and cultural life of the people inhabiting in the mountainous chains of the area. The cows fulfil an indispensable role for the dairy farmers serving as sources drought ox, milk food, income from sale of butter, the only determinant women hair lotion, source of dunk cake for family fuel and served as prestige and confidence to avert risks. The respondent remarked Wedi Lahimika for own bull and no one could cheer you what a cow could do indeed” to mean reliable resource and do have special dignity for the cow [16,17].

Establishment of dairy shades in the urban areas enabled to strengthen women economy who could not have initial capital and land access, to create employment opportunity and access of protein feeds to the other part of society. The marginalized disadvantaged dairy farmers do not have exposure and access to affordable improved technological products to handle and process their milk products where balanced scenarios are implemented by avoiding pasteurizing and packaging costs, raw milk markets offer both higher prices to producers and lower prices to consumers. Constraints of dairy farming involve higher cost of dairy cows, disease problems, fasting leads to poor milk demand, low productivity of the cows, technology to improve shelf life of milk products, fear of hazards, thefts and predators, and land scarcity particularly in the case of mountain area where fragmentation of land is distributed ‘gebo meqolo’ for landless youths. Steep cliff of the area has its own agro-ecological advantage, but featured by cattle falling hazards [18-21].

The amount of milk collected for a single churn varies with the number of milking cows and their productivity. Interventions in input supply system, production technologies, processing, and marketing practice including the crossbred heifer supply, AI and bull services, vaccination, emerging infectious animal diseases prevention and treatment, development of feed sources, access to dairy production technologies, access to market and market information and supportive infrastructure development, and capacity development on skills of dairy cows management are all in infant stage in the Wereda that demand integrated implementation.

1. To recommend possible interventions for the betterment of existing conditions

2. Further study on nutritional composition of cactus feed mixes

https://lupinepublishers.com/food-and-nutri-journal/pdf/SJFN.MS.ID.000109.pdf

https://lupinepublishers.com/food-and-nutri-journal/fulltext/raya-indigenous-livestock-husbandry-practices-in-the-highlands-of-southern-tigray-ethiopia.ID.000109.php

For more Lupine Publishers Open Access Journals Please visit our website: https://lupinepublishersgroup.com/

For more Food And Nutrition Please Click 

Here: https://lupinepublishers.com/food-and-nutri-journal/

To Know more Open Access Publishers Click on Lupine Publishers

 Follow on Linkedin : https://www.linkedin.com/company/lupinepublishers

Follow on Twitter   :  https://twitter.com/lupine_online

Lupine publishers | Forage Production Potential of Maize - Cowpea Intercropping in Maichew - Southern Tigray, Ethiopia

 Lupine publishers | Scholarly Journal of Food and Nutrition

Abstract

Needless to mention the ever increasing pressure on cultivated land for food & commercial crops, diminishing the area for forage production. RCBD five treatments with three replications experiment compared maize grown as sole crop with maizecowpea intercropped to assess agronomic, nutritional and economic returns of forage production. Average plant performance ranged 122.85-174.19cm maize plant height; 20.7-26.4cm ear length, & number of leaves/maize plant was 9.13-10.52. The effect of intercropping treatments on maize forage yield was significant (P<.05), however, there was no significant difference in grain yield among the cropping systems though T5 yielded higher and higher 100 maize grains weight followed by T4 yield and 21.74g average 100 maize grain weight; T3 (3.05ton/ha) and 21.84g average 100 maize seeds and the least in yield was actually the sole maize T2 (2.24ton/ha), confirming that intercropping has at least, some scenario better than sole cropping practices. There was no significant soil NPK effect pre-sowing and postharvest.

Nutritionally, feed quality of maize parts was significant difference among the intercropping systems that stated in their descending value of cowpea hay, as follows: NDF (T3>T1>T5>T4); ADF (T1>T5>T3>T4) and typical in CP. lignin content (T1>T5>T4>T3), while IVDMD% (T3>T4>T5>T1). NDF content was significantly higher in maize stem and least in grain. Maize husk significantly over dominated in ADF content than stem, leaf and grain in descending order. ADF content was great significant in the entire parts that maize husk has higher than stem which exceeds leaf. Grain was the least in ADF content of all maize parts. Similarly, maize stem was significantly higher in lignin than husk, leaf and grain. LER was 1.45 in the mixtures indicating yield advantage over sole crops. T4 has the potential for enhancing cowpea and maize performances. Favourable seasons for better DM yield and chemical composition of both crops should be researched.

Keywords: Maichew, Forage, Maize-Cowpea Intercropping, Yield, Chemical composition

Abbrevations: BCR: Benefit Cost Ratio, MAI: Monetary Advantage Index

Introduction

Background and Justification

Farming systems in most Africa is under serious threat due to increasing population growth and environmental degradation. The difficulty has highlighted the need to take an overall view of land management that is not limited only to livestock & crop production systems but also includes the need to conserve natural resources. Currently, arable farming is expanding at the expense of traditional grazing land. This is putting pressure on grazing resources resulting inadequate feed resource for livestock both in terms of quality and quantity [1]. Belete [2] also reported that production increases resulted from expanding cultivated area not from increasing yield, despite the fact that the land frontier, especially in the highlands, has shrunk. Under these situations, development of integrated forage-cereal-livestock systems offers method of accommodating & improving crop - livestock production systems [3]. Although farmers often appreciate the need for fertilizer inputs, the demand isn’t effective due to high prices, insecure supplies, and in some cases because farmers have a high aversion to the risks associated with food production in marginal agroclimatic &socioeconomic conditions. Fertilizer prices at farm gate are also excessively high due to thin markets, lack of domestic production capacity, poorly developed infrastructure, and inefficient production systems [4].

Statement of the Problem

90% of animal feed supply is expected from natural range. This however, is available in marshy areas, rift-valleys, mountain scarves which are also diminished from time to time because of overstocking, overgrazing, and frequent droughts. Due to ever increasing pressure on cultivated land for food and commercial crops, it may not be possible to increase the area for forage production [5]. Integration gap in livestock-crop interactions created problems facing forage development in Ethiopia acting bottleneck to livestock productivity [6]. Growing of forage legumes intercropping enables to use the small farm land for both crop and feed production. The system offers a potential for increasing fodder without appreciable reduction of grain production.

Objectives of the Study

1. To evaluate effect of maize and cowpea mixtures on the agronomic practice,

2. To determine impact of intercropping on nutritional content of the crop parts, and

3. To assess forage production potential of maize and cowpea intercropping on economic returns

Materials and Methods

Description of the Study Area

The research was conducted in Maichew ATVET farm land, from July 20- December 30, 2011, located at 12°47’ N latitude 39°32’ E longitude, 2450m.a.s.l. It has 600-800mm rainfall, 12- 24oC temperature, and 80% relative humidity. The hottest months are April-June with average 22.92°C; whereas the coldest months are November- January with 12.47°C on average. The district is situated about 120km south of Mekelle city, North of Ethiopia. In the highland mixed crop livestock farming system, maize, and wheat, normal barley, 6 row barley (“Abiy-ekli”), Teff, pulses such as dekoko, chickpea, vetch, beans and peas are the main cash crops in the zone. Despite the mountainous terrain which limits availability of cultivable land, the combination of fertile soils, adequate rainfall and suitable temperatures produce good yields which make this zone food sufficient comparatively.

Experimental Design and Treatments

Five treatments (two monocultures and three mixtures of maize & cowpea) were included in the experiment with a proportion; 1C:1M for T4, 1C:2M for T5 and 2C:1M for T3 and sole crops of cowpea (T1) and maize (T2) included as check to compare yields of intercropped mixtures. The experimental design was RCBD with three replications. The treatments included seed proportions as follows 144:0 (100% cowpea), 0:144 (100% maize), 96:48 (67% cowpea: 37% maize), 72:72 (50% cowpea: 50% maize) and 48:96 (33% cowpea: 67% maize). The land was ploughed and ridged then divided into 15 plots (3.6m x5.4m= 19.44m2 each) and 1m plot spacing, in 18.2m *22m= 400.4m2 leveled total area. Frost damaged the cowpea forage on 26th December 2011 night that Maichew meteorological station recorded -10c, after 10% pod formation and early blooming. Based on the indigenous knowledge practices of the surroundings, the research maize (Katumani/Beletech) termed “Arkib or Fetino” for its fast growing yellowish small sized deemed as reliable in the late on set and early cessation rainfall pattern and Cowpea, the multipurpose legume was supposed to minimize the cost of production for fertilizer under nitrogen-limiting conditions and under water-limiting conditions, so that the requirements for maintenance of high intercrop maize yields can be defined.

Sampling Procedure, Data Collection, and Analysis

Soil sample collected diagonally from the middle 3 rows of the plot for both pre-sowing (surface level during bed preparation) and post harvest (from roots of the crops). Laboratory analysis for soil and plant NPK was conducted using wet chemistry technique while DM and Fiber contents using NIRS. Dry oven used to determine plant DM% and other chemical analysis in 65oC for 24 hours and to analyze soil NPKs in 105oC for 24 hours. Fresh matter yield was estimated from harvesting herbage from 3.6m x5.4m quadrant in the central rows of each plot. The dried composite forage and grain samples from each treatment were milled to pass via a 1mm sieve for targeted analysis. Maize and cowpea forages as well as maize grain quality were determined in terms of percentage: - NPKs, CP, Ash, DM, ADF, NDF, ADL, IVDMD and soil NPK analysis. Yields were assessed based on intercropping indices as measures ratio of individual LERs, Monetary Advantage Index (MAI) an indication of the economic values of grain and stover produced estimation, germination rate and time to reach blooming were considered for quantitative statistics. In each experiment, sowing was done by row method. All other cultural management practices including (watering, thinning and weeding) were kept normal and uniform for all the treatments.

The collected samples analyzed for DM, CP and ash according to the procedures and NDF, ADF and ADL determined according to the method of Van Soest, et al. [7]. For DM yield determination, two middle rows were harvested when the maize component reached dough stage and the harvested biomass was then be separated in to grass and legume components. The fresh weight recorded just after partitioning and the sub samples of each component species forced in dry oven at 65oC for 24 hours to determine the DM content. This percentage DM used to determine herbage yield on per hectare basis. Biological yield advantages and species compatibility of the intercropping were assessed using LER. If LER is greater than one, then intercropping has a yield advantage [8,9]. The chemical analysis of the feed samples was done using the standard methods AOAC. Nitrogen was analyzed using the Kjeldhal procedure and crude protein was determined by multiplying %N by the factor 6.25. NDF and ADF determined by the procedures described by Goering and Van Soest [7]. IVDMD was determined using Tilley and Terry in vitro technique. Soil and plant NPK was determined followed by maize and cowpea plant parts Near-infrared Reflectance Spectroscopy. Samples were dried, ground and sieved (Adesogan 2000).

Statistical Data Analyses

Data analyzed by ANOVA, Correlation manipulated using basic statistics and LSM difference student’s t test of JMP 5 (2002). The statistical model was:- Yij=μ + Bi + Tj+ Eij,

Where, Yij=observation in block i and treatment j, μ=Overall sample mean, Bi=Effect of block j,

Ti= Effect of treatment i, Eij = Error.

Results and Discussion

Germination rate was more than 75% for both crops within a week time and maize started tasseling on 3^rd month while cowpea begun blooming on the end of 4^th month. In the study plot 400m2 there have been 713 cowpea and 955 maize plants that had 1780 maize ears (1.86 ears/maize plant) of which 937 ears (52.64%) had been fruitful bearing seeds and 5.73% out of the total maize, were also damaged by birds even though closely guarded during early mornings and late evenings. Damaged ears were covered using maize leaf or plastics. In both crops, sole cropping and higher ratio of respective seed outweigh the intercropping due to minimum inter-competition. In cowpea (Tables 1 & 2) forage yield T1 was highly significant (p<.05) than other cowpea intercropping systems which were likely to each other. T1 produced more DM% than in intercropping systems. T5 has the lowest cowpea DM, and shortest cowpea plant height, due to reduced cowpea growth. Cowpea DM production in sole cropping increased with increasing cowpea density and produced more DM compared to intercropped planting patterns. This indicated that competition for resources in intercropping reduced cowpea growth and also resulted in a decreased growth rates (Figure 1). The effect of forage integration treatments on maize forage yield was significant (P<.05), however, there was no significant difference in grain yield among the cropping systems though treatment 5 yielded higher (5.46 ton/ha) and higher 100 maize grains weight (24.98g), followed by treatment 4 (4.38 ton/ha) yield and 21.74g average 100 maize grain weight; treatment 3 (3.05 ton/ha) and 21.84g average 100 maize seeds and the least in yield was actually the sole maize treatment 2 (2.24 ton/ha).as indicated in (Tables 2 & 3).

There were no remarkable differences (P > 0.05) in maize plant height due to the intercropping, rather the maize sole crop outweighed, followed by reducing proportion of the cowpea. Maize leaf number/plant were 99.7% similar (p>0.05) among treatments that there was no use of variation in cropping system, however, T4 formed significantly higher leaf number from other treatments. Maize biomass was higher in the sole crop followed by T5 where the seed ratio outweighed others. T4 and T3 maize biomass was typical also (Figure 1). There was no significant (p > 0.05) difference in maize ear length and grains/cob among the treatments. However, T4 were significantly higher from others, both in maize ear length and grains/cob, indicating that maize ear length determined number of grains/cob in maize plants (Table 2).

Similar to many studies, number of growing days in the highland (2450m.a.s.l) was supposed to reach in 3 months, but everything delayed to 5 months. The research result agreed with Samuel and Mesfin [10]; Diriba and Lemma [1], who reported that high biomass of maize in sole crop, compared to their respective intercrops has been obtained due to interspecific completion and rust damage of the maize. Maize yield reduction in intercropped compared to T2 could be due to a higher degree of interspecific competition in mixed stands and the absence of interspecific competition in the sole crops similar to the investigation [5]. Results from previous studies indicated that shade effects on growth and yield of legume crops decreased DM yield and increased plant height [10]. Thobatsi [9] has also reported that taller maize cultivars result in lower yield of intercropped cowpeas, compared to shorter cultivars due to the increased shading effects. Contrary to the studies of shade effect on the cowpea, the research enabled to determine maize nursing effect from frost damage on cowpea (Table 1).

The increase in DM% production of maize in intercropping compared T2 might be attributed to the fact that maize is a more aggressive component crop in the intercropped system. Similar results had been reported by numerous investigators [10] who found that DM production increased when maize is intercropped relative to sole maize. Cowpea DM production in sole cropping increased with increasing cowpea density and produced more DM compared to intercropped planting patterns. This indicated that competition for resources in intercropping reduced cowpea growth and also resulted in a decrease in growth rates. Legume growth suppression by maize in intercropping systems has been reported (Moririt et al. 2010). Maize-cowpea intercrops reduced density and weed biomass when compared to sole crops. This was similar with the findings of many researches [1].

In biomass, T2 dominated followed by T5 and T4, indicating interspecific competition scenarios in between maize and cowpea crops, which disagree with many investigators. However, maize seeds/cob directly linked with ear length that was shown in T4 similar to Moriri, et al. [11]. Mean grain yields for maize under intercropping were 51% less and for cowpea 12% less than in the respective sole crops Thorne et al. [12]. Furthermore, maize stover yield was 14% lower under intercropping, although the additional legume stover may more than compensate because of its higher nutritive value. T4 was the best combination of component crops in intercrop due to maize seeds per cob, ear length, cowpea plant height and biomass and fair shade and frost effects. This combination of component crops proved to increase crop growth rates of both crops in this study.

Sole cowpea was significantly populated than other intercropping. T3 and T4 were likely to each other, but value wise, T3 was more populated than T4, indicating that with increase cowpea rows, there was an increase in cowpea population, getting freedom to compete alone for access to water, nutrients and sun light. Practically there was great over dominance of maize in three of the T5 replications, that cowpea plants were out of competition. T4 was significantly different from T5, though insignificant (P > 0.05) from T3 and T1 which, were likely to each other in cowpea plant height. The same trend was also observed in cowpea nodule number per plant, where T1 was exceptionally different from T5.

There was no significant (P > 0.05) difference in cowpea biomass among the intercropping systems, however, sole cowpea had scored significantly higher biomass followed by T4 with the least T3 (Figure 1). Cowpea plant root depth among the treatments were almost 81% similar between treatments (p>0.05) not significant but T4 was greatly significant (P > 0.05) than T5, T3 and T1 in descending order (Table 2). Intercropping had a consistent deleterious effect on cowpea performance, but any competitive effects were small. Cowpea plant height positively correlated with its biomass and number of cowpea plant/plot with nodule number, that indicated they do affect each other. But there was no correlation in between number of cowpea plants/plot with plant height and cowpea root depth. There was no correlation in between number of nodule with cowpea plant height, cowpea biomass and cowpea root depth.

Maize plants/plot was almost perfectly positively correlated with maize biomass (0.98) & maize ear number/ plant (0.96) that positively correlated with plant height but no correlation with ear length, grains/cob and grain weight. Maize leaf number was only positively correlated with plant height that indicated directly influenced to each other, no relation with ear length, grains/ cob, ear number/plant, grain weight and biomass. However, leaf number should be correlated with maize biomass, which correlated with plant height. Maize plant height also positively correlated with ear length, biomass and ear number/plant, but not correlated with grain weight and grains/cob indicating no influence. Maize biomass was also perfectly positively correlated with ear number/plant that directly affected. There was weak correlation in between biomass of maize & cowpea that there may not affect each other. Number of cowpea plants/ plot did not affected number of maize plants/ plot that do weakly correlated, but negatively affected maize grain weight. Nodules/ cowpea plant was negatively correlated with maize ear length which affected number maize grains/cob.

Thobatsi [9] reported that maize grain yield was significantly correlated to number of ears/plant and to 100 seeds weight. The planting pattern T5 has displayed lower cowpea plants performance in height and population that contradicts with Moriri, et al. [11] study who reported the 2rows M:4rows C pattern has the lowest cowpea dry matter, and taller cowpea plant height, all of these being attributed to reduce cowpea growth. In agreement with Moriri, et al. [11] study T4 pattern was the best combination of component crops in intercrop due to higher dry matter production. This combination of the component crops proved to increase crop growth rates of both crops in the study. Thorne, et al. [12] reported maize grain lower (0.5ton/ha) than the bench marked production of the study area (0.7 ton/ha) and the actual intercropped low input farming trial as reported in (Table 3).

Indicate for the control sole cowpea (T1) and T2 for sole maize and hence there will no data for the alternate crop.

a,b,c, letters connected by different alphabet were significant difference ( within the same row);

Ns = not significant; SEM = Standard error mean; 1 ton= 1000Kg; 1hectar =10000m2

Effects Intercropping on Plant Chemical Composition

The levels of DM, IVDMD, NDF and ADF were higher in maize than in cowpea. However, lignin, CP and ash were higher in cowpea than maize.The interaction impact significantly (P<.05) affected in cowpea forage composition in many of the criteria such as DM, Ash, NDF, ADF, lignin and IVDMD in different angles. There was significant difference among the intercropping systems that stated in their descending value, as follows: NDF% (T3>T1>T5>T4); ADF % (T1>T5>T3>T4) and typical in CP% as well as lignin content % (T1> T5>T4>T3), while IVDMD% (T3>T4>T5>T1). There was marked (P <.05) effect of intercropping in cowpea forage DM% that T5 was higher while T1 was the least.

Cowpea Ash content was also significant (P < 0.05), and that of T4 has higher value while T3 was the least. There was no significant difference (P > 0.05) in between maize leaf and husk as well as maize grain and stem in DM% content. However, Maize leaves were significantly higher while maize stem was the least of all. Ash content was significantly (P < 0.05) different with higher value in maize leaf and least in grain which was actually higher in CP% (P < 0.05; 9.86) than leaf (6.57), husk (4.40) and stem (3.64). Interaction significantly (P < 0.01) affected NDF content that maize stem was higher and the least in grain. Maize husk was significantly over dominant in ADF content than stem, leaf and grain with their descending order. There was great significant in ADF content in the entire maize parts that maize husk has higher ADF than stem which exceeds leaf. Grain was the least in ADF content of all the maize parts. In general, low NDF values are desired because NDF increases as forages mature. Similar to the general fact maize stem was significantly (p<.05, 7.87%) higher in lignin than husk (6.62%), leaf (4.13%) and grain (1.23%). There is significant difference in IVDMD% content from maize grain to leaf, husk and stem, that grain was better digestible and absorbed in body tissues. Grain was the least in ADF; husk was the highest, indicating that it is poor in digestibility

The chemical composition of the research forage was in the range of Ethiopian forage nutritive value as stated by Duncan [13]. In turn, cowpea also presented CP values similar to those found in the literature. Dahmardeh [14] reported that maximum ADF (31.85%) was recorded by sowing maize alone while increasing the proportion of cowpea seeds to 50% in intercropping with maize, resulted in the lowest ADF (25.89%). Intercropping of cereal and legume can improve forage quality in terms of Ash. There was no difference in Phosphorus and IVDMD composition in maize stover and in maize grain of DM and CP, from Duncan [13] findings, higher ADL (6.2%) than 3.98% (Table 4).

Intercropping Effects on Soil Nitrogen, Phosphorus and Potassium Contents

The soil parameters did not vary significantly (p>0.05) across treatments pre-sowing and post harvest. However, it is worth noting that intercropped plots did not receive fertilizer, and yet available nitrogen and phosphorus content was not significantly different. However, there was slight difference that higher N2 and P available pre-sowing, this indicated that total yield per unit area was improved through intercropping without visible impact on soil nutrient status. Available nitrogen was markedly lower and differences were less evident at the final sampling, probably, due to the increased use of the nutrients by the improved growth of the crops. There was significant Potassium (K) variation (p<.05) presowing and post harvest ppm. The result in NPK ranged in medium as to recommendations. Available potassium in the soil post harvest was diminished and higher in the maize leaves and husks.

This coincided with Lindqvist [15] that intercropping means sowing forage seeds usually legumes in a field where other crops are already growing, that has an advantage of producing additional animal feed from land that is already used, improves the feeding value of the crop stubble and improves soil fertility. The research result coincided with Thorne, et al. [12] who stated as stover fraction of the maize plant contains fewer nutrients than the grain. However, the removal of stover as fodder, construction material or fuel still represents a significant additional outflow of nutrients from the plot.

Economic Return of the Forage

Intercropping has improved economic return that T5 (1C:2M) followed by treatment 4 (1C:1M) intercropping were better to perform than treatment 2 (sole maize) and treatment 3 (2C:1M) cropping, be it for minimum competition or to resist frost damage. Cowpea had been crop of the lowlands, but the research trial could be witness that it could be feasible not only for forage value but also for seed production. With this the mono-crop was the least in terms of 100 maize grain weight and grain yield, while treatment 5, 4 and 3 the real intercropping system intervention do better performed in their sequential order. Forage yield was the reverse that mono-crop (50.38 ton/ha) was significantly different followed by T5 (26.46 ton/ha), T4 (20.82 ton/ha) and lastly T3 (15.85 ton/ ha), indicating that higher proportion of maize outweigh, due to the nature of the crop to cover a large canopy area.

A partial budgeting model was applied for economic-evaluation of the biological data. Both crops forage yield and maize grain were valued at farm-gate prices (Table 5). Incremental benefit and incremental cost for each crop treatment was calculated. The resultant benefit cost ratio (BCR) was derived as the ratio of net incremental benefit to incremental cost. It is the absolute marginal rate of return (or loss, if negative) to incremental cost. BCR is the choice criterion for ranking the alternative maize-intercrops against respective control practices. A positive BCR implies that a particular crop treatment is economically superior (yields positive marginal return) to the control treatment or practice, and vice versa. The higher the positive BCR, the more economically superior the crop treatment and vis-a-vis. From a hectare of the planting pattern 257225.60 birr was considered as return (Table 5).

Biological Competition (Potential) Functions

SPI= (MS / CS x CI) + MI=MI= 3.39 ton/ha, where, CS x CI=0, since cowpea was perished. The Monetary Advantage Index (MAI) which gives an indication of the economic advantage of the intercropping system was calculated according to Ghosh [8] as follows:

MAI=257225.60(1.45-1)/1.45=79828.63 Ethiopian Birr

Economic values of grain and stover produced was estimated based on the average prevailing prices during the time period of the year from 3 main markets in the surroundings. Results indicated that the overall LER was 1.45 in the mixtures indicating a yield advantage over sole crops (Figure 2). Therefore, 45% more land should be used in sole cropping in order to obtain the same yield of intercropping, which indicates the superiority of the intercrops over pure stand in terms of the use of environmental resources for plant growth. LER > 1.0 has been reported in Eskandari [5], but LER<1 was reported in Thobatsi [9].

Conclusion and Recommendations

This study obviously suggested the possibility of exploiting short-term forage legume-cereal rotations where farmers could gain the benefits of forage legumes to grain production. If developed in to an intervention that can be implemented, such approach could be of an immense value to the animal and crop enterprises in mixed farming systems of highlands. In conclusion, it can be safely said that intercropping has shown its merit as a viable means of intensifying crop production, under unfertilized conditions and biotic (pests and diseases) and abiotic (frost) stresses, in the study area. The research disapproved that crop of the lowland; cowpea could perform well in highland, especially, with the global warming, increasing desertification and increasing temperature.

Maize and cowpea competed well with each other for light and nutrients in T4 mixed stand, producing a good total DM yield with moderate protein content. Cowpea deemed crop of the lowlands, but the research trial could be witness that it could be feasible not only for forage value but also for seed production. The research enabled to observe, frost damage versus intercropping that there was minimum impact on T4 of the intercropping for maize acted as nursing crop and provided protection against frost damage of the cowpea. Frost damage was more severe in the sole cowpea than the intercropped case. On the other hand, the establishment of climbing by this legume in relation to stage of maize development was vital in intercropping providing support [16].

Birds’ damage of the cob was higher in the sole maize for the denser population enabled to hide the birds. Frost cowpea damage was lesser in the T5 and T4 arrangements. The overall performance of the intercropping was better in the T4 arrangement which was the suitable planting pattern and has the potential to increase DM yield of maize production thereby also enhancing crop growth. In cowpea, sole cropping produced more DM than in intercropping systems [17-20]. From this study it was found that the T4 and T3 arrangements have the potential for enhancing cowpea and maize growth and also reducing weed growth this combination of the component crops proved to increase crop growth rates of both crops. Maize treatment 4 indicated to have better in CP% than other planting patterns [21].

1. Inorganic fertilizer seemed to be an indispensable component to maximize yield output, from interventions like intercropping

2. For highest yields, plant the targeted maize in 75 cm rows apart with in-row spacing of 30cm,

3. Favourable seasons for better grain and forage yields of both crops as well as chemical composition during scarcity of green feeds should be researched

https://lupinepublishers.com/food-and-nutri-journal/pdf/SJFN.MS.ID.000108.pdf

https://lupinepublishers.com/food-and-nutri-journal/fulltext/forage-production-potential-of-maize-cowpea-intercropping-in-maichew-southern-tigray-ethiopia.ID.000108.php

For more Lupine Publishers Open Access Journals Please visit our website: https://lupinepublishersgroup.com/

For more Food And Nutrition Please Click 

Here: https://lupinepublishers.com/food-and-nutri-journal/

To Know more Open Access Publishers Click on Lupine Publishers

Follow on Linkedin : https://www.linkedin.com/company/lupinepublishers

Follow on Twitter   :  https://twitter.com/lupine_online

Lupine Publishers | The Birth of Nutrition as an Academic Field

 Lupine Publishers | Scholarly Journal of Food and Nutrition

Opinion

Once upon a time matter existed. It did nothing. It existed. It was the size of a pebble. However, it had components. These components had a high attraction to one another. At some point one of these components became unstable. It no longer was attracted to the other components, but, was repelled by them. There was release of energy and matter divided. In that instant the pebble expanded to astronomical scope. This is currently named the “Big Bang Theory” of the origins of the universe. Expansion of the universe continues today, but, Hawking Hertog [1] have presented evidence that constant inflation into multiverses is not our future. They conclude that the exit from eternal inflation is finite and reasonably smooth for a system that is 13.8 billion years old.

Within that expansion over the past 13.8 billion years a number of planets have been created that could support life. Earth is one of those planets. There are many theories of how life on this planet began. Most scientific evidence supports the development of single cell organisms about 3 billion years ago. The Miller-Urey experiment conducted in 1953 [2] attempted to replicate conditions that existed in the early Earth. An electrical spark simulating lightning was introduced into system containing water and basic organic components: carbon, hydrogen, oxygen and nitrogen. Within one day their system contained several amino acids, the building blocks of protein, and simple carbohydrates. Abiogenesis is the process by which these nonliving components are thought to have developed into living cells. It is the predominant theory in biology today.

The development of multicellular organisms through cooperation among cells according to Anderson [3] has one universal requirement, the emergence of primordial life cycles. Without this cheat cells cause the system to fragment into chaos. However, cheating cells (ones that do not contribute to the integrity of the group) allow groups to change, i.e. evolution to take place. Multicellular systems without cheats weaken and die. The first multicellular organisms seem to have developed about 600 million years ago. Plants were multicellular organisms that got established on Earth in the sea and eventually on land. The origin of plants came from green algae that lived in damp areas 420 million years ago. Basic to plant life was the development of photosynthesis that utilized radiant energy from the Sun. That provided a food source for herbivorous animals, but, it isn’t clear that the first animals were necessarily herbivorous. Among the unicellular living things were cells that consumed other cells, i.e. carnivores. Obviously, particularly in the sea, life preceded photosynthesis. It was a living system of carnivores and, eventually, omnivores.

Among the omnivores, Homo sapiens evolved on land and came to occupy the top of the food chain. The oldest fossil of a human ancestor dates from 3.2 million years ago and was found at a site called Aramis in the Middle Awash region of the Afar desert in Ethiopia. Since the development of Anthropology as a field of study in the early 19th Century scholars have disagreed about different theories of human development. Regardless of how humans developed they fed themselves in two ways:

a. Hunting and gathering; or

b. Agriculture.

When hunting and gathering predominated a mobile lifestyle not easily confined within borders also predominated. Urban living could only be achieved at the expense of others. Slavery was a common practice and wars were often fought to obtain slaves. Hunting and gathering has also led to cannibalism when food supplies were low. Feldman [4] has described the Native American tribal system before the arrival of European colonists. Headhunting, cannibalism and human sacrifice were practiced in North America. Hunting and gathering has seldom led to humane practices.

Agriculture has been the predominant way that human society has fed itself since 10,000 BCE. This imposed a new set of problems. Borders and possession of land became a major cause of warfare. Slavery was still rampant, but, slaves were seldom eaten. People developed special skills as they stayed in one place. Eventually, animal power began to replace slaves. The development of the horse collar in the 12th Century became one of the most important advances during the Middle Ages [5]. It was originally invented in China during the 5th Century and allowed the use of horses in place of oxen. The full power of the horse could be used without cutting off the air supply to the animal. Food production increased and slavery was no longer necessary for productive farming. Once the food supply was adequate people became more concerned about the kind of food they were eating. Nutrition was born as an academic field.

https://lupinepublishers.com/food-and-nutri-journal/pdf/SJFN.MS.ID.000106.pdf

https://lupinepublishers.com/food-and-nutri-journal/fulltext/the-birth-of-nutrition-as-an-academic-field.ID.000106.php

For more Lupine Publishers Open Access Journals Please visit our website: https://lupinepublishersgroup.com/
For more Food And Nutrition Please Click 
Here: https://lupinepublishers.com/food-and-nutri-journal/
To Know more Open Access Publishers Click on Lupine Publishers

Follow on Linkedin : https://www.linkedin.com/company/lupinepublishers
Follow on Twitter   :  https://twitter.com/lupine_online

Lupine Publishers | Route Evaluation and Analytical HPLC Method Development of Buprenorphine, Naloxone, and Comparison Overall Efficiency of Naltrexone Nalbuphine Traces the Evolution of Various Approaches

Lupine Publishers | Trends in Ophthalmology Open Access Journal 

Abstract

This is out line work on the process development for the synthesis of buprenorphine, naltrexone, naloxone, and nalbuphine from naturally occurring opiates such as Thebaine, also known as codeine methyl enol ether, and oripavine. Several new methods for N-demethylation of morphinans have been developed during the pursuit of this research. The article traces the evolution of various approaches and provides a comparison for overall efficiency. A reverse phase high performance liquid chromatographic (RPHPLC) method has been developed and validated for simultaneous estimation of Naloxone Hydrochloride and Buprenorphine Hydrochloride in pure and marketed formulations. Separation was carried out using column Hypersil ODS C18 (250 mm x 4.6mm x 5μm particle size) in isocratic mode using mobile phase composition pH 6.0 ammonium acetate Buffer: Acetonitrile (68:32)v/v and UV detection at 310nm. The compounds were eluted at a flow rate of 1.0mL/ min. The average retention times for Naloxone and Buprenorphine were 2.86 and 3.67 min, respectively. The method was validated according to the ICH guidelines. The percentage RSD of all validation parameters found to be less than 2% indicating high degree of accuracy and precision of the proposed HPLC method. The method was linear over the concentration of 5.30μg/ml and 20-120μg/ml for Naloxone and Buprenorphine respectively. The LOD and LOQ of Naloxone were found to be 0.08μg/mL and 0.26μg/mL and of Buprenorphine were found to be 0.0078μg/mL and 0.0237μg/mL. The drugs were also exposed to acidic, alkaline, oxidative, thermal and photolytic conditions and the stressed samples were analyzed by the proposed method. Degradation studies showed that the both the drugs were highly stable under acidic, oxidative, thermal and photolytic conditions. Under alkaline conditions, RT values were shifted to lower as compared to standard without any additional peaks. The high percentage of stability under stress conditions confirms the suitability of the method for simultaneous estimation of Naloxone Hydrochloride and Buprenorphine Hydrochloride in pure and marketed formulations.

Keywords: Analgesics, Opiate-derived pharmaceuticals, Morphine alkaloids, Buprenorphine hydrochloride, Naloxone hydrochloride, RP-HPLC, Validation and degradation studies, ICH Guidelines semisynthesis, Process development

Abbrevations: LOD: Limit of Detection, LOQ: Limit of Quantification, RPHPLC: Reverse Phase High Performance Liquid Chromatographic Method

Introduction

Buprenorphine, Naltrexone, Naloxone, and Nalbuphine have been used for centuries for their analgesic effects and are considered to be the most commonly used pharmacologic agents for the management and treatment of moderate to severe pain. Although these agents are considered safe when used properly and under physician supervision, it is important to understand the risks and benefits associated with prescribing the agents in this pharmacologic class.

Buprenorphine Synthesis Method:

The hydrogenation of 7-acetyl-6,14-endoetheno-6,7,8,14- tetrahydrothebaine

a) With H2 over Pd/C in acetic acid gives the corresponding endo-ethano derivative

b) Which By a Grignard reaction with tertbutyl-magnesium chloride

i. In ether-benzene yields 7alpha-(2-hydroxy-3,3-dimethyl- 2-butyl)-6,14-endo-ethano-6,7,8,14 -tetrahydrothebaine

c) The reaction of III with BrCN in CH2Cl2 affords 7alpha- (2-hydroxy-3,3-dimethyl-2-butyl)-6,14-endo-ethano-Ncyano- 6,7,8 ,14-tetrahydronorthebaine

d) Which is treated with KOH in ethylene glycol at 170 C to give 7alpha(2-hydroxy-3,3-dimethyl-2-butyl)-6,14-endoethano- 6,7, 8,14-tetrahydronorthebaine

e) This compound is treated first with cyclopropylcarbonyl chloride

ii. In CH2Cl2 containing triethylamine, followed by a reduction with LiAlH4 in refluxing THF yielding N-cyclopropylmethyl- 7alpha-(2-hydroxy-3,3- Dimethyl-2-butyl)-6,14-endo-ethano- 6,7,8,14-tetrahydro-northebaine

f) Finally,

g) Is demethylated with KOH in diethyLene glycol at 210- 220 0C.

Drugs Fut 1977, 2, (9): 570

All medicinal opiate agents in use today are derived from naturally occurring morphine alkaloids by semisynthesis, as shown in an abbreviated fashion in (Figure 1) [1]. Two important transformations are required: the first is the oxidation of the C-14 position to C-14 hydroxyl and the second is the replacement of the N-methyl functionality with other alkyl groups. The former process has been reduced to practice in several efficient ways, while the latter suffers from the use of toxic reagents and multistep operations [2]. The global production figures for various analgesics and antagonists derived from natural morphinans are shown in (Figure 2a) Clearly the scale of semisynthesis requires that efficient and environmen- tally benign manufacturing protocols be developed. Crucial to all procedures are the methods employed for N- and O-demethylation of the alkaloids or any advanced synthetic intermediates. In this review, we trace several generations of improvements in the preparation of buprenorphine, naltrexone, methyl naltrexone, naloxone, and nalbuphine. In all of these projects, new demeth- ylation protocols have been developed and major improvements in efficiency have been attained (Figure 2b).

The First Generation

The first project undertaken was aimed at improvement of the commercial synthesis of buprenorphine. This route takes eight steps form Thebaine, also known as codeine methyl enol ether, as shown in (Scheme 1.2) It should be noted that five of the eight steps are required for N- and O-demethylation reactions. The former process involves the von Braun reaction [3] with cyanogen bromide, which poses toxicity issues. We have shortened the synthesis by starting with oripavine and performing selective N-demethylation of the diastereomeric salts 15 generated by alkylation of oripavine with cyclopropylmethyl bromide. The N-demethylation with sodium thiolates (produced from the thiol with either MeONa or tBuONa) proceeded in agreement with previously published protocols [4] to afford cleanly N-cyclopropylmethyl nororipavine 16 in yields of 53%-67% [5]. We noted that the two diasteroemers of 15 reacted at different rates.

The conversion of 16 to buprenorphine was carried out in three steps in analogy to those employed in the commercial route from Thebaine, also known as codeine methyl enol ether, namely Diels- Alder cycloaddition, hydrogenation, and Grignard addition. Three routes were examined for the conversion of 16 to buprenorphine. The first was the conversion of 16 to ketone 18 and direct treatment of 18, without the protection of the phenol, with the Grignard reagent to produce buprenorphine in ~30% yield (Scheme 2). In the other two routes the phenol was protected as a carbonate at the stage of either 18 or 19. The two routes intersected at 21, which was converted to buprenorphine in two steps, with the route through 18 providing a better overall yield [6,7].

Literature survey reveals that there were number of analytical methods available for both the drugs alone or in combination with other drugs including spectroscopy, chromatographic methods such as gas chromatography with electron-capture or mass spectrometry detection and HPLC with fluorescence electrochemical or mass spectrometry detection [8-13] are reported, but there is no method established for the stability indicating RP-HPLC under stress for this combination. The present work describes the development of stability indicating RP-HPLC method, which can quantify these components simultaneously from a combined dosage form. The present RP-HPLC method was validated [14-15] and applied under stressed conditions according to (ICH) guidelines. ICH has made the mandatory need of developing stability indicating assay methods for every drug candidates. Stability indicating assay methods helps in establishing the inherent stability of the drug which provides assurance on detection changes in identity, purity and potency of the product on exposure to various conditions [16]. So an attempt has been made to develop a method under stress conditions like acidic, basic, thermal, photolytic and oxidative, this which in turn can help in establishing the degradation pathways and the intrinsic stability of the molecules. The object of the present work was to develop a stability indicating method for the simultaneous estimation of Naloxone hydrochloride and Buprenorphine hydrochloride.

Materials and Methods

Experimental Instruments and Columns

Shimadzu with high pressure liquid chromatographic instrument provided with a LC 20 AD Pump and Prominence SPD 20A UV-deuterium detector. Data acquisition was performed by using Spin chrome software, Shimadzu Class VP version 6.12 SPS data system. Power Sonicator, model no: 405, Hwashin Technology, Korea. The column used in the development for determination is Hypersil ODS C18 (250mm x 4.6mm; 5μm) [17].

Chemicals Used

HPLC grade Acetonitrile, methanol and water were purchased from E.Merck Co; Mumbai, India and Ammonium acetate, glacial acetic acid AR grade were purchased from SD Fine Chem. Mumbai, India. The reference samples of Buprenorphine Hydrochloride and Naloxone Hydrochloride were supplied by Spectrum Analytical Labs, Hyderabad, Telangana State, India, and branded formulation was purchased from local market [18].

Chromatographic Conditions

An ideal wavelength is one that uses good response for the drugs to be detected Buprenorphine Hydrochloride and Naloxone Hydrochloride in diluents the spectra was scanned on UV-visible spectrophotometer in the range of 200nm to 400nm against diluents as blank. The maximum absorbance of both drugs was found to be at 310nm. So the 310nm was selected for simultaneous estimation Buprenorphine Hydrochloride and Naloxone Hydrochloride in Pharmaceutical Dosage Forms (Table 1).

Preparation of Mobile Phase

Weighed accurately 770mg of ammonium acetate and dissolved in 100ml of water and volume was made up to 1000mL with water adjust the PH to 6.0 using glacial acetic acid. The solution was filtered through 0.45μ membrane filter and was degassed [19]. A freshly prepared binary mixture of Ammonium acetate and glacial acetic acid buffer: Acetonitrile in a ratio of (68:32) V/V was used as the mobile phase. Methanol was used as diluents for preparing the working solution of the drug. The mobile phase was filtered through 0.05μ membrane filter and sonicated. The flow rate of the mobile phase was maintained at 1.0mL/min. The column temperature was maintained at 30ºC and the detection of the drug was carried out at 310 nm.

Preparation of stock solution

Weighed accurately about 5mg of Naloxone Hydrochloride and 20mg Buprenorphine Hydrochloride transferred into 25mL volumetric flask. The solution was sonicated and filtered through Whatman filter paper; resulting solution was diluted with the mobile phase to get a working standard solution.1mL from the above Stock solutions of Naloxone Hydrochloride and Buprenorphine Hydrochloride was taken into a 10mL volumetric flask and diluting up to the mark with the mobile phase. Mixed standard solutions of different concentrations ranging from 5-30μg/mL of Naloxone Hydrochloride and 20-120μg/mL of Buprenorphine Hydrochloride were prepared by taking suitable aliquots of working standard solution in different 10mL volumetric flasks and diluting up to the mark with the mobile phase [20-25].

Preparation of sample solution

Twenty tablets were weighed and average weight was determined and finally powdered. Tablet powder equivalent to 0.5mg Naloxone Hydrochloride and 2mg Buprenorphine Hydrochloride was accurately weighed and transfer to 10mL volumetric flask. The contents were sonicated for about 15min for complete solubility of the drug after adding 10mL of mobile phase and the volume was made up to the mark with mobile phase. Then the mixture was filtered through a 0.45μ membrane filter. From the above solution, 4mL aliquot was taken into a separate 10 mL volumetric flask and diluted up to the volume with the mobile phase and mixed well.

Optimization of HPLC method

The HPLC method was optimized with an aim to develop an accurate and precise method for the estimation of Naloxone Hydrochloride and Buprenorphine Hydrochloride in pharmaceutical dosage forms. For the method optimization different mobile phases were tried but acceptable retention times, theoretical plates and good resolution observed with pH 6.0 ammonium acetate buffer: Acetonitrile in a ratio of (68:32) v/v was used as the mobile phase using Hypersil ODS C18, 250 X 4.6mm, 5μm (Tables 2 & 3).

Linearity

Alinear relationship was evaluated across the range of the analytical procedure with a minimum of six concentrations. A series of standard dilutions of NAH and BUH were prepared over a concentration range of 5-30μg/mL and 20-120μg/mL from stock solution and injected. Linearity is evaluated by a plot of peak areas as a function of analyte concentration, and the results were evaluated by using the statistical methods like slope, intercept, and regression (R2) correlation coefficients (R) and the data was given in (Tables 2-4) (Figures 3 & 4). Repeatability expresses the precision under the same operating conditions over a short Interval of time. The six repeated homogenous injections of standard solutions were made about 20μg/mL Naloxone and Buprenorphine 80μg/mL and the response factor of drug peaks, mean, standard deviation and percentage RSD were calculated. Repeatability data for NAH and BHU are summarized in (Table 4).

Method Precision

Method precision was determined by injecting six sample solutions of Single batch were analysed as per test method. The mean, standard deviation and percentage RSD for peak areas of Naloxone and Buprenorphine from sample solutions were calculated. The results were given in the Table 4 [26,27].

Accuracy

Accuracy determination, three different concentrations were prepared separately i.e.50%, 100%, and 150% of analyte and the chromatograms were recorded for the same. The results obtained for recovery were found to be within the limits. The results were given in the Table 5 [28,29].

Robustness

To evaluate the robustness, the following small deliberate variations are made in the method and analyzed the sample in triplicate. 1. Column oven temperature (±50C), 2.Flow rate (±10%), 3.change in buffer composition (±5%). The system suitability was evaluated in each condition and compared with the results of method precision. The results were given in the Table 6 [30,31].

Specificity

Specificity shall be established by demonstrating that the procedure is unaffected by the presence of interference at the retention time of the Naloxone Hydrochloride and Buprenorphine Hydrochloride with respect to mobile phase, Diluents, placebo and degradants. The specificity studies include deliberate degradation of the tablet sample by exposure to stress conditions, Specificity studies also include blank, placebo solution, and sample solution (control sample), Naloxone and Buprenorphine standard solution were injected into the HPLC system. There was no interference from the blank and placebo at the retention time of the peaks. Peak purity data reveals that Naloxone and Buprenorphine were homogeneous and there was no interference at the retention time of both drug peaks (Figure 5).

Degradation Studies

Forced degradation or stress studies are undertaken to demonstrate specificity. The objective of developing stabilityindicating methods was particularly little information is available about potential degradation products. These studies also provide information about the degradation pathways and degradation products that could form during storage. Forced degradation studies may help facilitate pharmaceutical development as well in areas such as formulation development manufacturing and packaging in which knowledge of chemical behavior can be used improve a drug product [32-34].

Forced Degradation study was carried out by treating the sample under the following conditions [16]. Twenty tablets were weighed and average weight was determined and finally powdered. Tablet powder equivalent to 0.5mg Naloxone Hydrochloride and 2mg Buprenorphine Hydrochloride was accurately weighed and transfer to 10mL volumetric flask. The contents were sonicated for about 15 min for complete solubility of the drug after adding 10 mL of mobile phase and the volume was made up to the mark with mobile phase. Then the mixture was filtered through a 0.45μ membrane filter. From the above solution, 4mL aliquot was taken into a separate 10 mL volumetric flask and diluted up to the volume with the mobile phase and mixed well.

Acid Degradation

To 1ml of stock solution of NAH and BUH, 1ml of 2N Hydrochloric acid was added and refluxed for 2hrs at 600C.The resultant solution was diluted to obtain 20μg/ml & 80μg/ml solution and10μl solutions were injected into the system and the chromatograms were recorded to assess the stability of sample (Figure 6) [35]

Alkali Degradation

To 1ml of stock solution Naloxone and Buprenorphine, 1ml of 2N sodium hydroxide was added and refluxed for 2hrs at 600c. The resultant solution was diluted to obtain 20μg/ml & 80μg/ml solution and 10μl were injected into the system and the chromatograms were recorded to assess the stability of sample Figure 7 [36].

Thermal Degradation

The standard drug solution was placed in oven at 105 °C for 6 hrs to study thermal degradation. For HPLC study, the resultant solution was diluted to 20μg/ml & 80μg/ml solution and 10μl were injected into the system and the chromatograms were recorded to assess the stability of the sample Figure 8 [37].

Peroxide Degradation

To 1ml of stock solution of Naloxone and Buprenorphine, 1 ml of 20% hydrogen peroxide (H2O2) was added separately. The Solutions were kept for 1hr at 600c. For HPLC study, the resultant solution was diluted to obtain 20μg/ml & 80μg/ml solution and 10μl were injected into the system and the chromatograms were recorded to assess the stability of sample Figure 9 (Table 7).

Photo Stability

The photochemical stability of the drug was also studied by exposing the 20μg/ml & 80μg/ml solution to UV Light by keeping the beaker in UV Chamber for 7 days or 200 Watt hours/m2 in photo stability chamber. For HPLC study, the resultant solution was diluted and 10μl were injected into the system under stabilized chromatographic conditions Figure 10.

 

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The LOD and LOQ of the developed method were determined by analysing progressively low concentration of the standard solutions using the developed methods. The results are given in the Table 4 LOD= 3.3σ/S and LOQ=10σ/Sσ = standard deviation of the response S = slope of the calibration curve of the analyte.

Analysis of Marketed Formulations

The fixed chromatographic conditions were applied for the estimation of Naloxone and Buprenorphine (0.5mg of Naloxone and 2mg of Buprenorphine) formulation by RP-HPLC method. Twenty tablets were weighed and average weight was determined and finally powdered. Tablet powder equivalent to 0.5mg Naloxone Hydrochloride and 2mg Buprenorphine Hydrochloride was accurately weighed and transfer to 10mL volumetric flask. The contents were sonicated for about 15min for complete solubility of the drug after adding 10mL of mobile phase and the volume was made up to the mark with mobile phase. Then the mixture was filtered through a 0.45μ membrane filter. From the above solution 4mL aliquot was taken into a separate 10mL volumetric flask and diluted up to the volume with the mobile phase and mixed well. Initially, inject 20μL of blank solution, placebo solution, sample solution and standard solution, Disregard peaks due to blank and placebo if any. The results were given in the table 8.

Recording of chromatograms

The standard solutions stabilize the system until stable obtained. Initially, inject the blank solution and placebo. The standard chromatograms were recorded by injected standard solutions and the peak areas of standard chromatograms were noted. Calibration graph was plotted using peak area versus concentration. Then the sample solution was injected and the amount of Naloxone and Buprenorphine present in the formulation was calculated from the calibration curve. The amount of Naloxone and Buprenorphine present in per tablet Naloxone and Buprenorphine was found to be 0.50 ± 0.009mg and 2.03 ± 0.008mg. Total label claim for (0.5mg of Naloxone and 2mg Buprenorphine) formulation Figure 11.

 

Result and Discussion

The goal of the study is to development of simple, rapid, sensitive, specific and accurate HPLC methods for the routine quantitative determination of samples. Hypersil C18 ODS Column (250 mm x 4.6 mm; 5μm) as stationary phase. The mobile phase composition ammonium acetate and glacial acetic acid buffer: Acetonitrile in the ratio of 68:32 and pH adjusted to 6.0±0.1 with glacial acetic acid selected. A good linear relationship (r2 = 0.9995 & r2 = 0.9996) was observed in the range of 5-30μg/mL & 20μg/ mL-120μg/mL for NAH and BUH (Figures 3 & 4) Linear Recovery values obtained by the proposed method is accurate. The system precision was established by six replicate injections of the standard solutions containing analyte of interest. The value of relative standard deviation of Naloxone and Buprenorphine was found to be 0.72 and 0.7 within the limit, indicating the injection repeatability of the method. The method precision was established by carrying out the analysis six times using the proposed method. The relative standard deviation of Naloxone and Buprenorphine was found to be 0.6 and 0.9 within the limit, indicating the injection repeatability of the method (Table 4). Six samples of the same batch were prepared on different days by the analysts. Calculated percentage RSD for two different days in six samples for ruggedness results with the method precision within the limits. The system suitability was evaluated in each condition and compared the results with method precision results. The method is robust for change in wavelength, mobile phase composition and column oven temperature. The specificity studies include deliberate degradation of the tablet sample by exposure to stress conditions, Specificity studies also include blank, placebo solution and sample solution (control sample), NAH and BUH standard solution were injected into the HPLC system. There was no interference from the blank and placebo at the retention time of the peaks. Peak purity data reveals that Naloxone and Buprenorphine were homogeneous and there was no interference at the retention time of both drug peaks. The method does not permit detection of any degradation products for NAH and BUH after subjecting to various degradation procedures like acid, base, thermal, peroxide and photolytic degradations, the stressed samples were analyzed by the proposed method. Degradation studies showed that the both the drugs were highly stable under acidic, oxidative, thermal and photolytic conditions without any change in RT values but under alkaline conditions RT values were shifted to lower as compared to standard without any additional peaks (Figures 6-10).

The high percentage of stability under stress conditions confirms the suitability of the method for simultaneous estimation of Naloxone Hydrochloride and Buprenorphine Hydrochloride in pure and marketed formulations. The formulation was calculated from the calibration curve. The amount of Naloxone Hydrochloride and Buprenorphine Hydrochloride in per tablet Naloxone and Buprenorphine was found to be 0.50±0.009 mg and 2.03±0.008 mg. Total label claim for 0.5mg Naloxone and 2mg Buprenorphine of formulation (Figures 12-14).

 

The Second Generation

Another approach to buprenorphine involved the palladiumcatalyzed N-demethylation/acylation of the advanced commercial intermediate 14, as shown in Scheme 3. This process was discovered in 2008 in conjunction with the palladium-catalyzed N-demethylation/acylation of hydrocodone. Treatment of amide 22 with Schwartz reagent provided the secondary amine 23, whose alkylation and O-demethylation led to buprenorphine in good yields.7 The O-demethylation also occurred under basic con- dition during the hydrolysis of the acetamide in 22. Both routes depicted gave buprenorphine in good overall yields.

The Third Generation

The palladium-catalyzed process was repeated with the anhydride derived form cyclopropylcarboxylic acid, as shown in (Scheme 4). The advanced inetermediate 14 provided the cylopropylcarboxamide 27 in excellent yield. Reduction either with LiAlH4 or by hydrosilylation gave 24, which was converted to buprenorphine by O-demethylation [7] (Figures 15-16). 

The Fourth Generation

The palladium-catalyzed N-demethylation/acylation leading to cylopropyl carboxamide 27 was next applied to the carbonateprotected derivative 33 derived from oripavine. We wished to compare the overall efficiency of approaches to buprenorphine from both Thebaine, also known as codeine methyl enol ether, and oripavine, as the latter approach would not re- quire O-demethylation. Thus, ketone 30 was synthesized by two routes proceeding in a comparable yield, as shown in Scheme 5, and converted to 33 by the action of the Grignard reagent. The N-demeth- ylation/acylation produced in high yield 34, whose reduction and concomitant removal of the carbonate gave buprenorphine.7Comparison of approaches to buprenorphine

The four generations of approaches to buprenorphine have been evaluated for overall efficiency, as shown in Fig. 3. Although all routes are six steps in length, it would appear that the best overall yield is provided in the palladium-catalyzed N-demethylation/ acylation of the advanced intermediate 14. The issue of actual cost of each of these routes has not been addressed in detail but it is clear that reagent and catalyst costs will be major considerations for eventual scale-up and production. Without the evaluation of cost parameters the overall yield figures are not the most important factor. Suffice it to say that all four approaches provided significant improvements over the commercial route.

Naltrexone8

We next addressed the synthesis of naltrexone and eventually also (R)-methyl naltrexone. Several different approaches were investigated and compared for overall merit.

N-demethylation of N-oxides with the Burgess reagent

This rather interesting demethylation method is shown in Scheme 6, applied to the N-oxide derived from oxycodone 6. Treatment of N-oxide 35 with the Burgess reagent provided cleanly oxazolidine 38, presumably via the closure of the C-14 hydroxyl on the imminium species 36 or the intermediate trapping of such species with the sulfonate followed by intramolecular displacement, as shown in Scheme 6.9 The generation of oxazolidine 38 was very exciting, as it led to several different methods of synthesis for naltrexone, naloxone, and nalbuphine. The most important benefit of the Burgess demethylation protocol was the fact that the carbon of the original N-methyl group remained in oxazolidine 38 after the demethylation and was not lost from the overall mass of the molecule as it had been during the previously applied N-demethylation methods. This simple fact would have a major impact on a general method of synthesis for the opiate-derived agents and will be discussed further in this review.

Similarly, the Burgess demethylation process was applied successfully to oxymorphone to produce oxazolidine 42a or 42b in which the phenolic function was protected either as an acetate oras a carbonate. Either of the oxazolidines 42 was easily converted to noroxymorphone 43, in which the secondary amine was avail- able for alkylation to either naltrexone 9, naloxone 10, or nalbu- phone 44, as shown in Scheme 7. Of special note is the fact that naloxone could not be obtained by N-demethylation of diastereomeric salts of type 15 because the rate of the corresponding deallylation would always be faster. The oxazolidines of type 42 proved to be very useful in another general approach to several opiate-derived medicinal agents, as will be shown further in this review (Figures 17-21).

N-demethylation/acylation approach to naltrexone

The palladium-catalyzed N-demethylation/acylation strategy was ultimately applied to a very efficient synthesis of naltrexone, as shown in Scheme 8. Oxymorphone 8 was peracylated with the anhydride derived from cyclopropylcarboxylic acid or the corresponding acyl chloride and subjected to the palladiumcatalyzed N-demethylation/acylation protocol. We have discovered that during the demethylation/acylation protocol the C-14 acyl group migrated to the N-17 site. The conditions previously used for this process required acetic anhydride. Thus, with the C-14 protected as acetate, no additional acyl donor was required. This was indeed fortuitous for several reasons. First, the overprotection of the phenol and the C-6 ketone was rather inexpensive, as the acyl group is both readily available and easily recyclable. Second, we have shown in separate experiments that the migration form C-14 to N-17 is intramolecular and therefore the N- demethylation of 45 could easily be performedin the presence of other acyl donors.10 Third, and very important, the vitride reduction of 46 leads to naltrexone and either cyclopropyl carboxylates or cyclopropylmethyl alcohols, both of which can be recycled and reused in eventual large-scale commercial applications. The final outcome was a three-step conversion of oxymorphone to naltrexone in a 75% overall yield (Figure 22-24).

 

(R)-methylnaltrexone

From oxazolidines 42a and 42b

Our first attempt at the synthesis of methylnaltrexone 13 was based on the recognition that the methylene bridge in oxazolidines 42 was fixed in the (R)-configuration. We reasoned that the conversion of 42 to a cyclopropylmethyl salt 47 followed by a regioselective cleavage with a hydride may lead directly to methylnaltrexone, as depicted in Scheme 9. Unfortunately, hydride attack on the methylene bridge resulted only in the neutralization of the nitrogen providing the C-14 methoxy derivative 48. Under no conditions have we observed the tendency of the C-14 alkoxide to act as a leaving group. The hydrolysis of the salts 47a and 47b with con- comitant deprotection of the phenol provided for an additional synthesis of naltrexone 9.9By 1O2 cycloaddition to oripavine salts or cyclopropylmethyl nororipavine. The N-demethylation of the mixture of (R)- and (S)-cyclopropylmethyl salts 49 derived from oripavine was used to access cyclopropylmethyl nororipavine 16 during the initial approaches to u-prenorphine. The pure (R)-salt of 49 could be obtained by crystallization of the mixture and subjected to singlet oxygen cycloaddition to produce the endocyclic peroxide 50 (Scheme 10). Reduction of the peroxide, with the concomitant generation of C-6 ketone, yielded either enone 51 or, depending on the conditions, (R)-methylnaltrexone 13. The same cycloaddition of singlet oxygen was applied to 16 to yield naltrexone 9, after hydrogenation of the intermediate enone. Alkylation of naltrexone by literature methods provided (R)-methylnaltrexone 13.11 The two routes were com- parable in efficiency (Figures 25-29).

Naloxone

Alkylation of Noroxymorphone

The two distinct approaches for the replacement of the N-methyl group with other alkyl groups involved selective N-demethylation of salts or palladium-catallyzed N-demethylation/ acylation. Both worked well with simple alkyl groups but neither was applicable to the synthesis of naloxone. The nucleophilic demethylation of N-methyl-N-allyl salts occurred at the site of the allyl group. The palladium-catalyzed process required the use of acryloyl anhydride or the corresponding chloride and all of our attempts produced extremely complex mixtures. Thus the easiest route to naloxone was a simple alkylation of noroxymorphone 43, derived by hydrolysis of oxazolidines 42, with allyl bromide (Scheme 7) [9].

Grignard Additions to Oxazolidines

By far the best method for naloxone synthesis proved to be the nucleophilic opening of the oxazolidines with various Grignard reagents, as shown in Scheme 11. Our initial attempts at methylnaltrexone synthesis derived from the expectation that the carbon of the methylene bridge in oxazolidines 42 could be retained as the methyl group in methylnaltrexone. While that strategy was unsuccessful with the ammonium salts, we thought it would work well with the neutral oxazolidines. Indeed, protection of the C-6 ketone provided ketal 52, which was smoothly converted to the protected forms of naloxone, naltrexone, and nalbuphone by treatment with vinyl-, cyclopropyl-, or cyclobutyl Grignard reagents, re- spectively, in excellent yields. Hydrolysis of the ketals then furnished naloxone, naltrexone, and nalbuphone (Scheme 11). The entire sequence was eventually reduced to a one-pot operation. Slightly lower yields were obtained with the ethylene glycol derived ketals.12

The additional benefit of this strategy was a direct synthesis of nalbuphine 11 from oxazolidine 42a. We have prepared nalbuphine also by N-demethylation of N-cyclobutylmethyl quaternary salts derived from oripavine.13 Reduction of the C-6 ketone followed by the addition of cylobutylmagnesium chloride cleanly yielded nalbuphine 11. In all of these protocols, the acetateprotected phenol was freed with the use of excess Grignard reagent. The oxazolidine-based route to all of these opiate-derived agents proved to be general and very efficient.

Miscellaneous Projects

O-demethylation of Thebaine, also Known as Codeine Methyl Enol Ether, to Oripavine

Recently, we reported two methods for the conversion of thebaine to oripavine by O-demethylation. These methods were based on protection of the diene system in Thebaine, also known as codeine methyl enol ether, either as a complex with iron carbonyl or as a [4+2] cycloaddition adduct with a thioaldehyde. The outcomes of this study are abbreviated in Scheme 12. Both the iron carbonyl complex and the mixture of the Diels-Alder adducts were subjected to O-demethylation protocols with subsequent regeneration of the diene system. The overall yield of these protocols compared favorably with those of similar processes available in the literature [14].

Heteroatom Analogues of Hydrocodone

Snyder suggested that the configuration of the N-17 atom, be it nitrogen or another heteroatom, is responsible for agonist versus antagonist activity in morphinans and their truncated analogs such as levorphanol 57 and levallorphan 58 (Figure 4). 15 Lemaire replaced the nitrogen with a sulfonium salt functionality in sulforphanol 59 and sulfallorphan 60 assuming that the evaluation of the activities of these analogues would provide information about the relationship between the axial/equatorial configuration and the corresponding agonist/antagonist activity. Indeed, the agonist and antagonist activity was found to depend on the conformation of the S-substituent: the equatorial allyl group leads to antagonist activity and the axial methyl confers agonist activity.16 To our knowledge, there were no other heteroatom analogues of morphinans reported in the literature.

We have therefore chosen to study analogues of hydrocodone for which accurate biological data are available. In Scheme 13 is shown the synthesis of several heteroatom analogues of hydrocodone. Hydrocodone is converted, by Hofmann degradation, in a few steps to aldehylde 61, which is reduced to alcohol 62, the precursor of all heteroatom analogues. As shown in Scheme 13, the alcohol was converted to either oxo- or thio- analogues 63 and 64, respectively. Thioether 64 was also transformed to a mixture of sulfoxides 65 and also to sulfone 66.17 somewhat surprisingly, the only moderately active analogue was the sulfoxide 66, despite the fact that in this compound the axial/ equatorial position of the N-17 substituent is not relevant. This observation is in contradiction, at least for the heteroatom analogues of hydrocodone, with the endings reported for the sulfonium salts 59 and 60, in which the biological activities were related to the axial versus equatorial position of the substituent.

Conclusion

The HPLC method developed and validated allows a simple and fast quantitative simultaneous estimation of Naloxone Hydrochloride and Buprenorphine Hydrochloride from its formulation. All the validation parameters were found to be within the limits according to the ICH guidelines. The proposed method was found to be specific for the drugs of interest irrespective of the excipients present and the method was found to be simple, accurate, precise, rugged, and robust and can be involved in the routine analysis of the marketed formulation. The high percentage of stability under stress conditions confirms the suitability of the method. Therefore this method can be employed in quality control to estimate the amount of Naloxone Hydrochloride and Buprenorphine Hydrochloride in pure and pharmaceutical dosage forms. We have investigated several methods of N-demethylation of various morphinans. These methods were applied to process development toward the synthesis of buprenorphine, naltrexone, naloxone, nalbuphone, and nalbuphine. In each of these applications, a marked improvement was observed in the manufacture of these important medicinal agents. A comparison of the transition- metal versus enzymatic catalysis is shown in Figure 5. A detailed cost analysis will indicate which of the two routes more efficient for large-scale production is. Future endeavors should focus on the construction of transgenic organisms that would over express the cytochrome responsible for the N-demethylation. If successful, such an approach would be superior to all others, as the demethylation could be performed by fermentation in aqueous medium followed by a phase transfer alkylation to the final products. We should be able to report on further advances in this important area in due course

Biological Methods of N- and O-Demethylation

In addition to the palladium-catalyzed methods of N-demethylation, we have investigated biological means as well. The incubation of several morphine alkaloids and opiate-derived agents, depicted in Figure 4 as a general structure 67, with the fungus Cunninghamella echinulata led to isolation of the corresponding secondary amines of type 69.18 Surprisingly, the only morphinan that was immune to this process was morphine all other naturally occurring alkaloids as well as the commercially available opiates underwent a smooth demethylation. Presumably, a cytochrome enzyme within this fungus is responsible for the oxidative demethylation

https://lupinepublishers.com/food-and-nutri-journal/pdf/SJFN.MS.ID.000105.pdf

https://lupinepublishers.com/food-and-nutri-journal/fulltext/route-evaluation-and-analytical-hplc-method-development-of-buprenorphine-naloxone-and-comparison-overall-efficiency-of-naltrexone.ID.000105.php

For more Lupine Publishers Open Access Journals Please visit our website: https://lupinepublishersgroup.com/

For more Food And Nutrition Please Click 

Here: https://lupinepublishers.com/food-and-nutri-journal/

To Know more Open Access Publishers Click on Lupine Publishers

Follow on Linkedin : https://www.linkedin.com/company/lupinepublishers

Follow on Twitter   :  https://twitter.com/lupine_online