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.