In Vivo Screening of the Extract of Solenostemon Monstachyus and Ocimum gratissimum for the Management of Type-2 Diabetes
Ikpesu TO* and Ezenwaka CO
Department of Biology, Federal University Otuoke, Nigeria
Submission: January 09, 2018; Published: July 16, 2018
*Corresponding author: Ikpesu TO, Department of Biology, Federal University Otuoke, Nigeria, Tel: +2348032312141; Email: firstname.lastname@example.org
How to cite this article: Ikpesu T, Ezenwaka C. In Vivo Screening of the Extract of Solenostemon Monstachyus and Ocimum gratissimum for the
Management of Type-2 Diabetes. Curre Res Diabetes & Obes J. 2018; 7(5): 555723. DOI: 10.19080/CRDOJ.2018.07.555723.
The efficacy of synergistic of Solenostemon monostachyus and Ocimum gratissimum for the management of type-2-diabetes using Zebra fish was investigated. The plants were dried, grounded and the powder was pooled together, homogenized and extracted for 7 hours using the Soxhlet apparatus. A total of 75 zebra fish were used and were divided into five groups of five juveniles per test concentration in three replicates. Group A was given de-chlorinated water; B was treated with 0.25mg/L diazinon only while other groups were exposed to the same concentration as in group B, but with different concentrations of the plant’s extract. Group C, D and E received 250, 500 and 750mg/L respectively. Water quality parameters and blood chemistry were estimated in the experimental and control fish on day 7, 14, 21 and 28th. All the blood parameters were significantly (<0.05) affected by diazinon when compared with the control. But were reversible in the plants extract mixture treated fish and were dose and time dependent reduction, even less than the control group. This finding had shown the efficacy of these plants’ extract and the use of zebra fish as toxicological tool. Thus, thorough study of these plants mixture is indispensable, so as to find a lasting solution to the ailment that has been given people a nightmare.
Keywords: Solenostemon monstachyus; Ocimum gratissimum; Mixture; Diazinon; Zebra fish; Human
Diabetes is one of the most noxious and precarious ailment that has been a menace to the life of people. The condition is mainly defined by the level of hyperglycemias which gives rise to the risk of micro-vascular damage . Diabetes mellitus is of two types; type I (insulin-dependent), this is when the insulin producing β-cells are not functioning well therefore no enough insulin production, and type II (noninsulin dependent) caused by relative insulin insufficiency due to insulin resistance-the inability of the insulin to tell the cells to use glucose in addition to insufficient insulin to overcome this resistance. Of a truth diabetes has robbed many people in the past years in living a healthy and normal life . The controls of diabetes depend on the triad of food, activity and insulin or oral agents. All these must be balanced properly by the diabetic patient.
It has been estimates by the World Health Organization (WHO) that over 366 Million people worldwide are affected by diabetes mellitus, and many without efficacious diabetes care . A recent revelation by the WHO indicates that diabetes has tripled in the last two decade globally with the highest
prevalence rates found in developing countries . The WHO
report indicates that in extreme cases, up to 30-50% of the adult population in some developing countries have been afflicted with diabetes . Chinenye  reported that in Nigeria, with the population of over 180 million people (2006 census), an estimated six million people have full blown diabetes mellitus.
The 1992 National prevalence study on Non-communicable diseases conducted by the Federal Ministry of Health in 13 states of the Federation indicated a prevalence rate of 2.7% with a prevalence of 2.6% in adult males and 2.8% in adult females. Reports from subsequent studies as hospital records indicated an alarming increase in both prevalence and incidence of diabetes among all ethnic groups and social classes in the world . Medicinal plants have been used in many years back to treat diseases all over the world. Some medicinal plants have been proven to be anti-diabetic plants used to treat diabetes mellitus by the local folks. Some of such plants are Solenostemon monostachyus (SM) and Ocimum gratissimum (OG), but have not been examined medically. Therefore, the study was designed to investigate the acclaimed anti-diabetic abilities of the extract mixture of Solenostemon monostachyus and Ocimum gratissimum extract macerated in acetone.
Diazinon (98.5%purity) that was used for fish bioassay
was obtained from chemical Service, West Chester, UK, Aquaria
(locally built), Aerators from Agro chemicals, Canada, ethyl
3-aminobenzoate methanesulfonate salt, Sigma.
The zebra fish used for the test were raised in the laboratory
in a well-aerated tarpaulin tank for three months. From the tank,
15 post juveniles’ fish weighing 23.20±0.12gm and 17.00±0.13cm
in length were taken into well-aerated containers, to avoid
hyperactivity, injuries and shock. The fish were examined for
any pathological sign and washed with 1% KMnO4 solution,
and transferred to glass aquaria (50x25x25cm) containing dechlorinated
tap water. They were allowed to acclimatize to their
new environment (glass aquaria) conditions for 14 days prior
to experiment and were fed with 30% protein pellets. Water
was replaced regularly and immunological parameters of the
water were kept within the recommended range with electrical
aerators. At the end of the acclimatization, all the fish were in
good health and no mortality observed.
A well-drained, sunny location within Federal University
Otuoke Nigeria premises earmarked for botanical gardens
was used for the cultivation of the herbs. The soil was
tested to determine the soil pH (6.0-6.8) and nutrient levels.
Geophysics Department of the University assist in ascertained
the physicochemical parameters of the soil. The seeds of S.
monostachyus and O. gratissimum were purchased from the
market and cultivated within 2 hours. The plants were monitored
and grew into maturity within two months.
The fresh leaves of O. gratissimum and S. monostachyus
were collected from the garden and dried separately at room
temperature (22+0.15) °C for 21 days in the laboratory. They
were grounded separately using Nakai blender and filtered
through a 40-mesh screen. They were grounded separately
using Nakai blender and filtered through a 40-mesh screen
Equal volume (1.5kg) each of the two plants powders and were
homogenized. The dry powder was extracted by maceration at
35-37 °C; five times for 18 to 20hrs with 70% ethanol. Filtered
under vacuum and dried using rotary evaporator (Heidolph,
Schwabach, Germany) at 35-40 °C. The extract was weighed and
preserved aseptically at 4 °C.
The photo-chemical compositions of the plants were
analyzed using the methods described by Trease & Evans .
The following chemical composition of the plants leaves were
determined quantitatively; Alkaloid, Terpene, Flavonoids,
Saponins, Anthraquinones, Tanin, Phlobatanins, Phenol and
Cardiac glycoside .
The fish were divided into five groups comprising five fish
per test concentration. They were exposed to the concentrations
of organophosphate that has been experimentally proven to
induce hyperglycemias , and other treatments as stated below.
Group A was given de-chlorinated tap water; B was treated
with 0.25mg/L diazinon only while other groups were treated
to the same concentration as in group B, but with different
concentrations of the plants extracts mixture. Group C, D and
E received 250, 500, and 750mg/L respectively. Water quality
parameters were kept within the normal range with the help of
aerator. The blood sugar was estimated in the experimental and
control fish on day 7, 14, 21 and 28th.
At the end of each experimental period (day 7, 14, 21
and 28th), a fish was removed from each aquarium and
immediately anesthetized with MS222 (ethyl 3-aminobenzoate
methanesulfonate salt, Sigma). Blood sample was taken by
puncturing the caudal vessels with a 20-gauge needle and
aspirating 0.2-0.4mL sample of mixed arterial and venous blood
into a heparinized syringe, a technique shown to minimize
dilution by tissue fluids . The blood sample was stored in
heparinized blood collecting duct for the estimation of blood
sugar level. The blood was allowed to clot for 30min, centrifuged
at 2000g for 15min for clear separation of the serum, and stored
at -80 °C until the analysis.
Folin-Wu protein free filtrate was prepared by adding nine
volumes of the mixture containing eight parts N/12 H2SO4 and
one part of sodium tong state directly to the sera separated from
the whole blood . The medium was filtered and the filtrate was
taken in a tube and two ml of alkaline copper sulphate solution
was added. The optical density was measured at 420nm in a
spectrophotometer. Simultaneously, standard glucose solution
containing 1mg•ml and a blank containing 2ml distilled water
was taken for the observations.
The data of the efficacy of synergistic of different
concentrations of S. monstachyus and O. gratissimum for the
management of type-2-diabetes were presented as mean±SD,
from 5 independent experiments with triplicates. The difference
between the control and the various treatments and within the
treatments were analyses using the student’s t- test at 95%
confidence level  and one-way analysis of variance SPSS
(14.0 version), SPSS Inc, Chicago, USA. P≤0.05 was considered
to be significant.
The water quality parameters (pH, temperature, dissolved
oxygen, alkalinity and hardness) monitored during the
investigation was not significantly different between various
concentrations of the pesticide and the control and within
concentrations (p>0.05) (Table 1).
A: Distilled water; B: 0.25mg/L diazinon treatment; C: 0.25mg/L diazinon and 250mg/L equal mixture of crude extract of S monostachyus and
O gratissimum; D: 0.25mg/L diazinon and 500mg/L equal mixture of crude extract of S monostachyus and O gratissimum; E: with 0.25mg/L
diazinon and 750mg/L equal mixture of crude extract of S monostachyus and O gratissimum.
The results of the photochemical composition of the
leaves extracts of the investigated plants showed the presents
of Alkaloid, Terpene, Flavonoids, Saponins, Deoxy-Sugar,
Anthraquinones, Tanin, Phlobatanins and Cardiac Glycoside.
The magnitude of concentrations of the chemicals in these plant
leaves extract differs as represented with depiction (Table 2).
Keys: *Present in moderately concentration; **Present in high concentrations; ≠ Not detected
The responses of the zebra fish to diazinon and to the
different concentrations of the synergistic of S. monostachyus
and O. gratissimum is shown in Figure 1. The glucose level in
aqueous treated fish (group A) shows no significant difference
between the days of exposure (p>0.05). However, in the diazinon
alone treatment, the fish were hyperglycaemic, which increases
with exposure duration and varies significantly (p<0.05). In
the treatment C, the fish responses is similar to B treated fish
but was not significant (p>0.05), though the effect of the plant
extract was slightly obvious. There was fluctuation in the D
treatment, although not significant (p>0.05), but the effects
was quite obvious and comparable to the control. E treated fish
shows hypoglycaemic as the day’s progresses. On day 7 and
14th, the sugar level is similar to that of the control, while day
21 and 28th, the level was lower than the control, the fish were
hypoglycaemic and the variation was highly significant (p<0.01).
Herbal medicines have played an important role in treating
diabetes in various parts of the world for centuries, and
their synergistic properties due to the presence of variety of
components within herbals extract are beneficial to multifactorial
diseases like diabetes . In this investigation, the
responses of the hyperglycaemic induced zebra fish to the
synergistic of S. monostachyus and O. gratissimum revealed
doses and time dependent recovery, with the concentration of
500mg/L plants extracts comparable with the control. When
the concentrations of the extracts was increased to 750mg/L
there was spontaneous decrease in the sugar level, far below the
reference value (Figure 1) and was highly significant (p<0.1) on
This anti-diabetic’s property S. monostachyus and O.
Gratissimum may be attributed to the synergistic and the
potency of their photochemical compositions. Alkaloid is known
to exhibits hypoglycaemic activity and antioxidant properties
[12,13]. Terpene often refers to as terpenoids has a broad range
of the biological properties, including cancer chemo preventive
effects, antimicrobial, antifungal, antiviral, ant hyperglycemic,
anti-inflammatory, and ant parasitic activities . Saponin is
essentially good in glycemic control as well as its principal roles
in preventing different metabolic disorders and liver damage
caused by hyperglycemias. Its anti-diabetic activity is attributed
to its components that plays vital activity in there leasing of
insulin from the pancreas that is, it exerts a direct insulin tropic
2-hydroxy-3-methyl-anthrqui¬none is another
photochemical component of the investigated plants and was
also identified as the hypoglycaemic active components of
binate . Although Anthraquinones glycosides have been
known to cause diarrhea, aglycone has several other biological
activities such as antibacterial and anti-inflammatory effects
. Similarly Ye et al.  reported that early intervention
with Anthraquinones glycosides significantly improved glucose
tolerance and restored early-phase insulin secretion in db/
db mice. Equally, the tannin photochemical observed in S.
monostachyus and O. gratissimum was also reported in extract
from the amaranth grain, finger millet, field bean, sunflower
seeds, drumstick, and amaranth leaves, and it exerted significant
higher antioxidant and anti-diabetic activities . Phenols are
known for their anti-hyperglycaemic activity , was present
in high concentrations in S. monostachyus and moderately
concentrated in O. gratissimum. This agrees with the local uses of
the leaves of the plant for the treatment of diabetes by traditional
medicine practitioners .
Diabetes mellitus is becoming a threat to world, with the
significant achievements in treatment modalities and preventive
measures; its prevalence has risen exponentially. Because of
these limitations, there is continuous need for new and more
effective therapies to combat the surge. The synergistic of S.
monostachyus and O. gratissimum had shown ameliorating effects
in the control of hyperglycemias and justify the traditional uses
of the plants for the treatment of diabetes.
Further study is necessary to investigate the mechanism
behind the hypoglycaemic effect of the synergistic of these two
plants. We strongly believe that they are promising anti-diabetic
constituents that will be helpful for the management of type 2
diabetes, and stabilize blood sugar level for longer per