CONCEPT REPORT By Linda Sakala A STUDY ON THE EFFECT OF EMINIA HOLUBII (MUNKOYO) ROOTS ON QUANTITY OF BREASTMILK IN LACTATING RATS INTRODUCTION Breast feeding is the best way to provide infants with the nutrition they need for healthy growth and development. (WHO and UNICEF,2018). WHO recommends it be commenced the first hour after birth then exclusively from then until six months. Complementary feeding is then recommended from 6 months up to 2 years and onwards. Only 36% infants are exclusively breastfed up to 6months to date worldwide With 3 million associated with infant mortality due to suboptimal breastfeeding in west and Central Africa (Infant and young child feeding, 2021). Therefore medical costs on the child’s health including productivity costs on economies are reduced if a child is breastfed optimally (Victora et al.,2016). Lactation failure due to insufficient start of lactation or reduced breastmilk production can lead to serious issues in the infant like reduced caloric intake, hypernatremia, dehydration or hyperbilirubinemia. (Wilde, 2021) Galactagogues or lactogenics may play a role in milk enhancement whose absence may contribute to a decrease in milk synthesis (Foong et al., 2021). Eminia holubii is a herb with trailing or climbing stems growing from a woody rootstock. E. Holubii (Munkoyo) has been believed to have galactagogue effects from anecdotal data (Mukuma Mwale, 2014) to the author’s knowledge no published information about E.Holubii having medicinal properties apart from as a beverage. But anecdotal data suggests its use as a galactagogue (Mukuma Mwale, 2014). Eminia holubii is a herb with trailing or climbing stems growing from a woody rootstock. The roots are dried and used to make the popular local beverage Munkoyo (as are the roots of certain species of Rhynchosia). (Eminia holubii (Hemsl.) Taub. — The Plant List, 2021). The Munkoyo root (Rhynchosia species) is from a sub shrub found as an under storey plant in the woodlands particularly the miombo woodlands. In Zambia it covers about 47% of the total land area of Zambia (Sekeli and Phiri, 2002). Consequently, the Munkoyo roots are found in different parts of the country. Munkoyo has been believed to have galactagogue effects from anecdotal data (Mukuma Mwale, 2014). There has been no published information(to the author’s knowledge) about Munkoyo root having medicinal properties other than as a beverage or cereal. Herbal galactagogues documented such as fenugreek have been shown in animal studies to possibly increase breastmilk by increasing the secretion of oxytocin and insulin (Sevrin et al., 2021). Some studies have been shown to support the fact that nutrition of the mother may play a major role in lactation. A study done by Ogwege et al showed that when vitamin c and alpha tocopherol were administered to wistar rats, levels of prolactin where increased supporting the theory that nutrition enhances breastmilk production (Okweje et al., 2019). This may perhaps be a possible way,besides increase in prolactin, in which the increase is breastmilk is achieved via the use of herbal galactagogues. LITERATURE REVIEW Galactagogues have been shown to exert some of their effects via dopamine receptors on the hormones oxytocin and prolactin (Oppong Bekoe et al., 2018). A study done in Luangwa district of Zambia by Tembo et al found only 16% of babies in the study population were exclusively breastfed up 6months. The prevalence of exclusive breastfeeding however was at about 61% as of 2015, slightly above the 60% target set for Zambia. With Zambia generally having a drop in exclusive breastfeeding of about 35% by 4-5months. The lack of it was met with reasons ranging from lack of support for the breastfeeding mothers and lack of knowledge of other benefits of breastfeeding (Tembo et al.,2015). The great body of evidence surrounds the use of especially domperidone and metoclopramide, with studies demonstrating moderate short-term improvements in breast milk supply (Grzeskowiak et al,2019). These are also met with adverse drug effects on the mother and infant such as cardiac arrythmias (prolonged QT), xerostomia in mothers with domperidone, depression with metoclopramide. (Wang et al., 2018) Whereas sulpiride and chlorpromazine may cause extrapyramidal side effects. Herbal galactagogues are increasingly being used in postpartum hypogalactia the most documented being fenugreek and blessed thistle (Liu et al., 2015). Fenugreek has been shown to influence the maintenance of lactation in ruminants (Oppong Bekoe et al., 2018). One study conducted in Ghana done by Ali et al found that as much as 83.8% of mothers in the study used galactagogues and 92% of those reported an increase in supply within 24hrs. The common herbs used by these mothers ranged from groundnut/peanut soup prepared with Bra leaves (Hibiscus sabdariffa), hot black tea, Werewere/Agushi (Citrulus colocynthis) prepared with Bra leaves, and Abemudro (a polyherbal formulation)(Ali et al., 2020). ).. one other study which was done by Husseinzadeh et al, also demonstrated the fact that the aqueous extract of Pimpinella anisum L. seeds traditionally used as galactagogue had milk enhancing effects. The method used was weighing the pups after a feed, and it was observed that the pups on the aqueous (1 g/kg) and ethanolic extracts (1 g/kg) increased the milk production significantly (p < 0.001), with about 68.1% and 81% more milk being produced, respectively, than in the control group (Hosseinzade et al., 2014). STATEMENT OF THE PROBLEM Adequate breastmilk is one of the mainstays for a child’s survival with breastfeeding being recommended to be exclusive the first 6months and complementary feeding for 2years and above thereafter(WHO,2018). Inadequate breast milk supply is a frequently reported reason for early discontinuation of breastfeeding and represents a critical opportunity for intervening to improve breastfeeding outcomes. Despite a lack of substantial evidence on true lactation failure,a look into galactagogues would play an important role in increasing breastmilk supply(Grzeskowiak et al,. 2019). Animal studies have been done on herbal galactagogues such as fenugreek which has shown to possibly increase breastmilk by increasing the secretion of oxytocin and insulin (Sevrin et al., 2021). The commonly used galactagogue worldwide has been domperidone (Grzeskowiak et al., 2019) whereas metoclopramide is commonly prescribed in our local setting. E. Holubii has anecdotally been used for years as a herbal galactagogue by Zambian locals with no documented evidence of its use as a galactagogue based on literature search (Mukuma Mwale, 2014). RESEARCH QUESTIONS RESEARCH QUESTIONS 1. Does Eminia holubii have breastmilk enhancing effects? 2. Do the pups whose dams are lactating show an increase in weight after suckling compared to the controls? 3. Which of the extracts show a significant change in weight of the pups? OBJECTIVES MAIN OBJECTIVE To determine if E.holubii enhances breastmilk secretion in lactating rats. SPECIFIC OBJECTIVES 1. To determine via HPLC the phytochemiocal constituents of E.Holubii 2. To acertain if there is a difference in weight in pups after suckling when E.holubii or distilled water are administered to the dams in the different groups. 3. To ascertain if there are any observed differences in the different extracts administered PREPARATION OF E.HOLUBII ROOTS The roots will be collected and then shadow-dried For the aqueous extract, the root powder (100 g) will be added to 1000 mL hot water, boiled for 15 minutes, and filtered through a cloth For the ethanolic extract, the root powders (100 g) will be defatted with petroleum ether (40–60°C) using the Soxhlet apparatus. Then, the powder will be macerated in 800 mL ethanol (80%, v/v) for 72 hours, and the mixture will be subsequently filtered and concentrated in vacuo at 40°C. The residue will be suspended in saline solution. ACUTE TOXICITY Different doses of the aqueous and ethanolic extracts of E. holubii will be administrated orally and intraperitoneally to groups of six mice. The number of deaths will be counted at 48 hours after the treatment. LD50 values and corresponding confidence limits will be determined by the Litchfield and Wilcoxon method (PHARM/PCS version 4). PHYTOCHEMICAL SCREENING. Phytochemical screening will be performed as indicated below using the procedures outlined by Trease (Trease G. E., 2002) and Sofowora (Sofowora A., 1993). 1. Test for Tannins: Two mls of aqueous extract will be shaken vigorously with 2 mls distilled water and few drops of ferric chloride (FeCl3) solution (0.1 M) were added. The formation of a green precipitate will be an indication of the presence of tannins (Trease G. E., 2002). 2. Test for Saponins: Five mls of aqueous extract will be shaken vigorously with 5 mls of distilled water in a test tube and will be warmed. The formation of a stable foam will be taken as an indication for the presence of saponins. (Sofowora A., 1993). 3. Test for flavonoids (Shinoda reagent test): One to two fragments of metallic Magnesium will be mixed with 3-4 mls extract, then 0.5 mls concentrated hydrochloric acid will be added; after some few minutes a red color will confim the presence of flavonols, orange- for the flavons, red-violaceous-characteristic to flavanones or green- in the case of flavanols (Trease G. E., 2002). 4. Liebermann-Burchard test for steroids and triterpenoides: Three to ten mls of hydroalcoholic extract will be evaporated in a porcelain capsule, the residue will be dissolved in 0.5 mls chloroform then 0.5 mls of acetic anhydride will be added. The resulting solution will be transferred to a dry test tube, followed by the addition of 1-2 mls concentrated Sulphuric acid to the test tube bottom; when sterols or triterpenoids are present in solution, a red-brown or violet ring appears after some minutes at the contact zone between the two liquids as well as a green-bluish or violet upper layer (Sofowora A., 1993). 5. Test for alkaloids: Three mls of aqueous extract will be stirred with 3 mls of 1 % HCl on a steam bath. Mayer’s reagent will be then added to the mixture. Turbidity of the resulting precipitate will be taken as evidence for the presence of alkaloids. (Sofowora A., 1993) 6. Test for cardiac glycosides (Keller-killani test): Two mls of the extract will be dissolved in 2 mls of glacial acetic acid containing one drop of FeCl3 solution. The mixture will be poured into a test tube containing 1 ml of concentrated sulphuric acid. A brown ring at the interphase indicates the presence of a deoxy sugar characteristic of cardenolides (Trease G. E., 2002). PROCEDURE OF ADMINISTRATION OF E HOLUBII TO RATS Female rats (6–8 weeks old, body weight of 200–250 g) will be obtained from Lusaka,Zambia. All procedures of animal care and experiments will be approved by the University of Zambia Biomedical ethics committee. The method is a modification of Lompo-Ouedraogo et al's method (Koko et al., 2019) 30 lactating dams (weighing 250 ± 25 g), which are at the starting of lactation and suckling four to six pups each, will be used for this test. Group 1 is to receive orally 0.5 mL of saline (control group), and groups 2–5 received orally the determined safe dosage per kg body weight of the aqueous and ethanolic extracts per 0.5 mL saline. All animals will be treated daily at 2:30 pm. The production of milk will be estimated 23 hours after gavage. Milk production will be measured from Day 3 to Day 15 of lactation. Milk yield and dams' weight, and weight gain of pups will be calculated every day. During the test time, the pups will be weighed daily at 8:30 am (w1) and then isolated from their mothers for 4 hours. At 12:30 pm, the pups will weighed (w2), returned to their mother, and permitted to feed for 1 hour. They will be weighed again (w3) at 1:30 pm. The milk yield 23 hours after gavage will be estimated as w3 – w2. The daily milk yield will be corrected for the loss of weight caused by the metabolic processes in the pups during the suckling period. The value used will be (w2 – w1)/4. Next, this value will be multiplied by the number of suckling hours per day and added to the daily suckling gain. Every day, gain in pups' weight will be measured from w2.
