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Effects of Polyherbal Formulation of Allium sativum and Persea americana Seeds' Extracts on Postprandial Hyperglycemia and Sucrose Digestion in Acute Treatment of Normoglycemic Rats.


Obesity and diabetes mellitus (DM) are among the most challenging metabolic disorders in the world. The clinical management of long-term complications associated to them is expensive for patients and governments [1]. Obesity and T2DM lead to reduction in both life expectancy and quality [2]. The global prevalence of DM in adults above 18 years was estimated at 422 million adults [3], Asia and Africa being the most at risk [4].

DM is a combination of heterogeneous disorders commonly presenting with glucose excursions and glucose intolerance, which arise because of derangements in the regulatory systems for storage and mobilization of metabolic fuels [2]. Hyperglycemia, one of the primary symptoms of T2D, gradually induces oxidative stress, insulin resistance, and impairment of insulin secretion [5]. Hyperglycemia, which can also arise from chronic overnutrition, is responsible for long-term complications such as macrovascular and microvascular damages [6,7]. Therefore, control of hyperglycemia, especially postprandial hyperglycemia, is critical in the treatment of diabetic patients and individuals with impaired glucose tolerance [8]. Unfortunately, conventional oral antihyperglycemic agents are not only expensive [6,9] but also have flatulence, diarrhea, abdominal bloating, nausea, headache, pancreatitis (rare), vomiting, sense of fullness, and hypoglycemia as side effects [10]. This justifies the growing interest toward combinations/polyherbal therapies used as alternative treatment with improved efficacy [11,12]. These include Andrographis paniculata and Gynura procumbens ethanolic extracts [13] and garlic and metformin [14].

Garlic is a member of the genus Allium. It is a spice belonging to the family of Alliaceae [15]. It possesses anticancer, antiviral, antioxidant, anti-inflammatory, and antidiabetic properties [15]. Avocado or Persea americana (P.a), another plant found in tropical and subtropical regions, belongs to the family Lauraceae [16]. Avocado seeds are used as natural antioxidants to treat gastrointestinal irregularities, anemia [17,18], arthritis, and hypertension [19,20].

Avocado leaf preparations have been used as long-term treatment of diabetes [21-23]. Moreover, in an acute treatment, they were shown to reduce blood glucose level after 5 h [24] to 6 h [25]. The timing could be improved with plant combinations, which offer multiple targets for treatment and may act via synergistic, antagonistic, or potentiating effects. This study was designed to study the effects of aqueous extract (AE) and hydroethanolic extract (HEE) of Allium sativum (A.s) and P. americana seeds solely or in combination (formulation) on postprandial hyperglycemia, sucrose digestion, and to compare the alkaloid content of both plant extracts.

Materials and Methods

All the chemicals used were of analytical grade and included ethanol, Fe[Cl.sub.3], HCl, and phenanthroline, purchased from Sigma Co., Louis, MO, USA.

Plant materials and preparation

Allium sativum and P.a fruits were bought from a local market in Buea (Cameroon) in February 2016. They were identified at the national herbarium as belonging to the families Alliaceae and Lauraceae, respectively. The peels of the A.s bulb were removed, and the bulb was shade dried in open air for one month. In addition, the P.a fruits were allowed to get ripe; the seeds were removed and were cut into small pieces. Then, they were shade dried in open air for one month. The dried materials were ground to obtain powder, from which the different extracts were prepared as previously described [26].

Preparation of aqueous extract

One hundred grams (100 g) of either A.s or P.a seeds were placed in 800 mL of water for maceration for 48 h. After filtration, the filtrate was evaporated at 50[degrees]C using an oven to obtain the AEs.

Preparation of hydroethanolic extract

One hundred grams (100 g) of either A.s or P.a seeds were placed in 800 mL of water/ethanol (95%) in a ratio of 1:1 (v/v) for maceration for 48 h. After filtration, the filtrate was evaporated at 50[degrees]C to obtain the HEEs.

Determination of alkaloid content

Alkaloid content was evaluated following the method reported by Singh et al. [27]. One hundred milligrams of the powder were mixed in 10 mL of ethanol (80%). The supernatant was used for the estimation of the total alkaloid content. The reaction mixture contained 1 mL of extract, 1 mL of 0.025 M Fe[Cl.sub.3] in 0.5 M HCl, and 1 mL of 0.05 M of 1,10-phenanthroline in ethanol. The mixture was incubated for 30 min using a water bath at the temperature of 70 [+ or -] 2[degrees]C. Absorbance of red colored complex developed was read at 510 nm against reagent blank. Alkaloid content was expressed as microgram ([micro]g) equivalence of quinine/milliliter (mL) of extract.

Animals and treatments

Thirty-five adult male albino Wistar rats weighing between 150 and 220 g were obtained from the Laboratory of Biochemistry, University of Yaounde I. The study protocol was approved by the institutional review board of the University of Yaounde 1. Animals were housed in clean with not more than five rats per cage and maintained under standard laboratory conditions (room temperature with dark/light cycle 12/12 h). They were fed with a standard diet and normal tap water ad libitum.

Study of antihyperglycemic activity: Oral glucose tolerance test

Oral glucose tolerance test (OGTT), an in vivo acute test was conducted as described by Al-Malki [28], to compare the effect of combination (200 mg/kg b.w HEE P.a + 200 mg/kg b.w. AE P.a), to individual constituent HEE P.a and AE A.s, in preventing the absorption and the rise of glycemia. After 12 h overnight fasting, blood glucose at baseline (T0) was measured from the tails of all the rats using a glucometer and test strips (One touch Gluco-plus). The rats were randomly distributed into the following groups:

Group 1: Positive control (PC) group was given water orally.

Group 2: Aqueous extract of Allium sativum (AEAs) (400 mg/kg body weight).

Group 3: Hydroethanolic extract of Allium sativum (HEEAs)

(400 mg/kg b.w.).

Group 4: Aqueous extract of Persea americana seeds (AEPa) (400 mg/kg b.w.).

Group 5: Hydroethanolic extract of Persea americana seeds (HEEPa) (400 mg/kg b. w).

Group 6: Glibenclamide (GB, 4 mg/kg b. w).

Group 7: Formulation 400 mg/kg b. w. (combination 200 mg AE of A. sativum and 200 mg of HEE of P. americana seeds). The combination included the poorer (HEE of P. americana) extract and the most efficacious (AE of A. sativum) extract. Thirty (30) minutes after administration of extracts, animals in all the groups were given an overload glucose solution (2 g/kg body weight). Blood glucose levels were determined with a glucometer by tail prick at 30, 60, 90, and 120 min after glucose loading. Administration of extracts and glucose was done orally in water 5 mL/Kg body weight. The dose of 400 mg/kg administered with P. americana once and A.s were reported not to be genotoxic [29] and hepatotoxic for animals [30].

Study of the effects of a formulation on in vivo digestion of sucrose

The protocol used was similar to OGTT with some modifications. The same combination at 400 mg/kg b.w. (200 mg/kg AE of A. sativum and 200 mg/kg HEE of P. americana) was administered, followed with 2 g/kg sucrose solution. Glibenclamide was replaced by acarbose (3 mg/kg b.w. as reference drugs). Administration of extracts and substrate was done orally at the same time to allow the interactions between extracts, substrates, and enzymes in the gastrointestinal track of the rats. Follow-up was done with blood glucose, measured as end products of sucrose digestion and indirect indicator of invertase activity.

Data processing and statistical analysis

The in vitro assays were done in triplicate. Results were expressed as mean [+ or -] standard deviation. Statistical Package for Social Sciences (SPSS 17.0 Chicago Inc.) for Windows was used to analyze data. Oneway analysis of variance followed by a post hoc test (Waller-Duncan) was used to compare continuous variables in different groups. The software GraphPad prism version 6.0 (GraphPad Prism, INC, CA, USA) was used to determine the area under the curve (AUC) by linear regression as expression of efficacy of the extracts. Results were considered significant at 95% confidence interval.


Determination of alkaloids content of different plant extracts

It was observed that the extract obtained from a mixture of water/ethanol (1:1) contains higher amount of alkaloids than water alone. In addition, HEE of P.a seeds contains eight folds of alkaloids than HEE of A.s (Table 1).

Glucose lowering potential of Allium sativum extracts on normoglycemic rats

It was observed that there was an increase of blood glucose (p < 0.05) in all the groups in the first 30 min of the experiment, followed by a decrease in all the groups (Figure 1).

Following adjustment from baseline (Figure 2), it was observed that, during the first 30 min, the PC group receiving (water + glucose) only had a similar profile with HEE of A.s: 70% increase from the baseline. Thereafter, blood glucose dropped for 40% in control (PC), while in HEE As400 group, the BG level remains higher than in all including the group receiving glibenclamide, a reference drug. Glibenclamide group (GB 3 mg/kg) showed the lowest increase all through the experiment with a regulation to baseline after 82 min. The efficacy of AE As400 in lowering BG was closely similar to glibenclamide (p > 0.05), making it the more efficacious extract to be used in the combination.

Glucose lowering potential of Persea americana seed extract on normoglycemic rats

It clearly appears that glibenclamide can prevent the increase in glucose absorption 30 min after administration, better than AE and HEE of P. americana. After 60 min, it was observed that all the tested groups exhibit blood glucose higher than reference and control groups. Only glibenclamide could regulate glucose increase after 80 min. HEE Pa400 was poorer in reducing blood glucose, making it part of the combination to be tested in Group 7 (Figure 3).

Effect of formulation (AE As200 + HEE Pa200 combination) of extracts on OGTT

Given that P.a and A.s extracts have been traditionally used to treat diabetes and other metabolic disorders, we then studied the effect of the most hyperglycemic extract of HEEPa and the most efficient or lowering extract of Allium sativum (AEAs).

Results of Figure 4 revealed that there is a glycemic peak in all the groups after 30 min with the highest peak associated with the positive control group (71 mg/dL, p > 0.05), the lowest peak associated with the reference group (21 mg/dL), and the group receiving the AE of A. sativum having the lowest peak (27 mg/dL) among the test groups. At time 60 min, the combination is observed to have the lowest peak (16

mg/dL), thus challenging the reference group. From time 60 min till the end of the experiment, both the reference group and the group receiving AE of A. sativum have peaks lower than the positive control; meanwhile, the HEE of P. americana has a peak higher than that of the positive control. In addition, it is only after 90 min that the combination presents a peak higher than that of the positive control (p < 0.05 at 2 h). It clearly appears that AE As200 potentiates the efficacy of HEE Pa200.

Efficacy of extracts on postprandial glycemia

From the above graphs, AUC, which provides a better expression of the efficacy of the extracts, was computed. Moreover, the result reveals that the combination has a lower AUC value compared to the positive control and the group receiving the HEE of P. americana. Thus, it is confirmed that, in the formulation, AE As potentiates or improves the effect of HEEPa by lowering the AUC value of the HEE of P. americana, although not comparable to the reference drug, glibenclamide (Figure 3).

Effect of combination of extracts on sucrose digestion

Thirty minutes after oral overload of sucrose solution, the blood glucose level in all the groups attained a peak. Then, the level progressively slows down over time (Figure 6). However, the highest peak was observed with the group receiving the combination (38.66 mg/dL), the lowest peak is associated with the reference group (12 mg/dL), and the group receiving the HEE P.a400 has the lowest peak among the test groups. During all the experiments, the group receiving the combination maintains the highest peak compared to the control and test groups. From 60 min till the end of the experiment, the AE As400 and the HEE Pa400 presented peaks higher than the reference-acarbose group (p < 0.05) and the positive control (p < 0.05).

Efficacy of extracts expressed as AUC revealed that both the individual extracts (AE A.s400: 1645; HEE P.a400 = 1315) and the Combination400 (AE A.s200 + HEE P.a200 = 2830 mg min/dL) had higher AUC values than the positive control (PC: 1273). However, the standard drug, acarbose, presented an AUC lower than the positive control (1273 vs 279.7 mg min/dL; p < 0.05), respectively (Figure 7).


Interest in functional foods is increasing daily in eating habits of many communities. Garlic is commonly used worldwide as spice and also for its medicinal virtue. This study showed in Figure 2 that aqueous extract (AEAs400) of garlic could efficiently reduce postprandial hyperglycemia within 2 h to level comparable to glibenclamide, a diabetic reference drug (Figures 2 and 4). This result is in accordance with many studies [23,31,32]. In the absence of extracts as observed in Figures 2-4, the increase of glycemic peak after 30 min, as also reported by Ullah et al. [7], represents situation where glucose is easily and directly absorbed by intestinal cells through glucose transporters (GLUT) in the intestine, mainly GLUT-2 [33]. In fact, after a meal intake, there is an increase in blood glucose levels that stimulates insulin secretion resulting in an increase in transportation, biotransformation, and storage in muscles and fat tissues [7] promoted by insulin release. This can explain results in positive control group (Figure 6). The presence of garlic extracts AEAs and HEEAs reduced postprandial hyperglycemia; therefore, they justify their use in the traditional treatment of type 2 diabetes [34]. The hyperglycemia lowering potential of garlic extracts (Figure 4) has been attributed to an active ingredient called thiosulfinates that stimulates insulin release from pancreas [28]. It probably reduced oxidative stress and regulated glucose uptake by peripheral insulin-sensitive tissues, thereby limiting progression toward complications associated to diabetes [35].

Avocado seeds have been considered for long time as waste products because of its high content of antinutrient constituents such as tannins, phytic acid, and alkaloids [36]. This study demonstrated that P. americana is very rich in alkaloids compared to A. sativum (Table 1). Alkaloids are known to enhance glucose-induced insulin [4] to induce relatively high glucose uptake and to possess good antioxidant potential at low dosages [37]. They could also activate PI3K/Akt and suppress PTP1B, protein tyrosine phosphatase, thereby promoting the synthesis of glycogen from glucose [38]. This can justify why aqueous extract AEPa400 was more efficient in reducing sugar than HEE Pa400 compared to positive control after 60 min (Figure 4). Previous studies on acute administration of pear leaves' extracts showed that effective time for glucose level regulation is only at 6 h after a single dose of the extract was administered, producing 60.02 [+ or -] 6.83% reduction in blood glucose level [25]. This could explain why in this study after 2 h the capacity of P. americana extracts to reduce hyper-postprandial glycaemia (HPPG) remains relatively low. The moderate activity of pear seeds can be attributed to bioactive molecules belonging to triterpenoids, tannins, flavonoids, saponins, and polyphenols [32,39,40]. Polyphenols like smeathxanthone A from Garcinia smeathmanii [41] and bioactive molecules' extracts from Nypa fruticans extracts [42] have been used to justify glucose lowering potentials.

Plant formulation involved combinations of extracts preparation for polyherbal therapy. It offers the advantage to have diverse pharmacological principles, capable to produce maximum therapeutic efficacy with minimum side effects [11,12].

In this study, formulation based on HEE Pa400 with the poorer efficacy (Figure 3) and AE As400 A.s with best efficacy showed that AEAs were capable to improve HEEPa efficacy after 2 h (Figure 4) instead of 6 h as reported with leaves. Thus, the combination of extracts ameliorates the activity of the extract of P. americana seeds, which alone showed only dampening effect on blood glucose level after 2 h. AEAs200 potentiates the efficacy of HEE Pa200 (Figure 5). Such improvement in efficacy function of time can prevent crisis often observed with patients like comas [43]. A combination A.s + P.s can better regulate high blood sugar than HEE Pa and reduce complications mediated by free radicals such as cardiovascular disease, retinopathy, nephropathy, neuropathy, leg ulcers, and gangrene [7,44,45]. A.s may improve the efficacy of P.a through complexity of bioactive molecules in the mixture on reducing free radicals in the pancreas, activation of [beta]-cells in the pancreas for insulin secretion [3], and increase the rate of glucose clearance from the blood stream, thereby reducing the rise in postprandial glycemia [4,46]. Many formulations of herbal extracts with improved efficacy on hyperglycemia have been previously reported with synergistic effects [13,47]. Other formulations between pure or herbal/pure molecules have been reported. These include metformin and rosiglitazone [47,48] and garlic and metformin [14].

Given the formulation based on (AEAs200 + HEE Pa200) potentiates the effects of HEEPa on postprandial glucose (Figure 5), there should be awareness when such formulation interacts with sucrose, a disaccharide found in diverse meals. Neither P. americana nor A. sativum extract could inhibit sucrose with efficacy comparable to acarbose (Figures 6 and 7). Instead, combination (1:1) of both showed activation of sucrase. Sucrose binds to the active site on sucrase, and this puts stress on the bond between the two sugars that make up sucrose. The bond breaks, thereby releasing glucose and fructose. The activation of enzyme by the combination contradicts the study by Malunga et al. [1] in which inhibition of digestive enzyme constitutes a therapeutic approach. Increased activity of sucrase triggered by combination can be due to the change brought in pH environment of Gastro-intestinal tract (GIT), promoting ion protonation or stimulating alkaline cations [49]. Such sucrase activation can be very dangerous for diabetic in critical situation such as comas. The use of formulations for diabetes treatment is on high demand [14,50]. However, care on good posology should then be taken when using polyherbal formulations.


Postprandial hyperglycemia remains a therapeutic target of interest in the management of DM and its associated complications. A.s AE at the dose 400 mg/kg b.w. lowered postprandial glucose better than HEE of P.a at 400 mg/kg b.w. In a formulation from the two plants (AEAs200 + HEE Pa400), A.s improves glucose lowering capacity of P.a seeds. However, the formulation instead activates sucrose digestion.

Authors' Declaration

There is no conflict of interest in this work.

Authors' Contributions

BGKA conceived, designed, and supervised the study and drafted the manuscript; WTK, GNT, EAT, and VMT prepared extracts and analyzed the data; JLN and JO supervised the work and corrected the manuscript.


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Boris Gabin Kingue Azantsa (1,2*), Wilfried Tchetmi Kuikoua (2), Guy Nguemto Takuissu (1), Erica Afuh- Mbi Takwi (2), Vanessa Maingain Tagne (2), Judith Laure Ngondi (1), Julius Oben (1)

(1) Laboratory of Nutrition and Nutritional Biochemistry, Department of Biochemistry, University of Yaounde 1, PO Box: 812, Yaounde, Cameroon

(2) Department of Biochemistry and Molecular Biology, University of Buea, PO Box: 63, Buea, Cameroon

(*) Corresponding author: Azantsa BGK, Laboratory of Nutrition and Nutritional Biochemistry, Department of Biochemistry, University of Yaounde 1, PO Box: 812, Yaounde, Cameroon, Tel: +237 677920184; E-mail:

Received: September 4, 2017; Accepted: March 07, 2018; Published: March 14, 2018

DOI: 10.4172/0974-8369.1000432
Table 1: Alkaloid content of extracts

Materials          Aqueous extract          Hydroethanolic extract

Allium sativum     1.10 [+ or -] 0.04 (a)    1.92 [+ or -] 0.13 (b)
Persea americana   9.77 [+ or -] 0.25 (c)   15.38 [+ or -] 0.17 (d)

Results are expressed as mean [+ or -] SD. Alkaloids are expressed in
equivalence of quinine. For each plant (lines) and each solvent
(column), variables with different letters a, b, c, and d associated
are significantly different at p < 0.05.
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Title Annotation:Research Article
Author:Azantsa, Boris Gabin Kingue; Kuikoua, Wilfried Tchetmi; Takuissu, Guy Nguemto; Takwi, Erica Afuh-Mbi
Publication:Biology and Medicine
Date:Mar 1, 2018
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