Int J Pharm Pharm Sci, Vol 12, Issue 7, 79-82Original Article

EFFECT OF COMBINATION OF AQUEOUS LEAF EXTRACTS OF PSIDIUM GUAJAVA LINN AND MORINGA OLEIFERA LAM ON DIABETES MELLITUS

MATHEW ANU JAYAMOL1*, REJI ASHLEY2, JOHNS NITHYA3, SHAMSUDEEN SHIJINA4

1*Department of Pharmaceutical Chemistry, Nirmala College of Pharmacy, Muvattupuzha, Kerala, India, 2,3,4Fourth Year B. Pharm, Nirmala College of Pharmacy, Muvattupuzha, Kerala, India
Email: anu.jayamol@gmail.com

Received: 26 Jan 2020, Revised and Accepted: 25 May 2020

ABSTRACT

Objective: To determine the anti-diabetic activity of combined aqueous extracts (1:1mixture) of dry leaves of Psidium guajava linn and Moringa oleifera lam as well as to compare the anti-diabetic activity of these plants by in vitro methods.

Methods: In vitro alpha amylase inhibitory assay was performed on porcine alpha amylase and the absorbance was measured at 540 nm using a microplate reader and glucose diffusion inhibitory assay using dialysis membrane. Acarbose was used as the standard in the above mentioned methods.

Results: The mixture (1:1) of aqueous plant extracts (at a concentration of 100µg/ml) of Psidium guajava linn and Moringa oleifera lam exhibited 72.08333% inhibition with IC50 value of 10.9µg/ml. The leaf extracts of Psidium guajava (at a concentration 100µg/ml) exhibited 71.23288% of a α amylase inhibitory activity with an IC50 values 19.883µg/ml whereas the leaf extracts of Moringa oleifera (at a concentration of 100µg/ml) exhibited 70.58824% of α amylase inhibitory activity with an IC50 value of 27.974 µg/ml. The Acarbose (standard drug) at a concentration of 100µg/ml showed 72.09302% inhibitory effect on the α amylase activity with an IC50 value 8.9µg/ml. In glucose diffusion inhibition assay the mixture of plant extracts exhibited 76.57% inhibition at 150 min which produces more effects than the two plants. The aqueous extract of Psidium guajava leaves exhibited maximum glucose diffusion inhibition (75.32%) at 150 min as well as Moringa oleifera leaf extract showed the maximum inhibition of 73.70% at the same time interval. For acarbose the percentage was 82.74 at 150 min. The interpretation of the results was done by one-way anova method.

Conclusion: The combined extract of the leaves of the 2 plants was found to be more effective than individual plant extracts against diabetes. On comparison of two plants Psidium guajava was found to be more active against diabetes than Moringa oleifera. Also the potentiation effect shown by the combination of extract may be due to synergistic effect of the phytochemical constituents. As the 1:1 mixture of the aqueous extract is found to be more active, the combination of the two plants can be used to formulate drugs for treating diabetes.

Keywords: Alpha amylase inhibitory assay, Glucose diffusion inhibition assay method, Psidium guajava, Moringa oleifera, and IC50

© 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2020v12i7.36823. Journal homepage: https://innovareacademics.in/journals/index.php/ijpps

INTRODUCTION

Psidium guajava Linn is a fruit-bearing tree commonly known as guava, goiaba, guayaba, goavier, perala which belongs to the family Myrtaceae (fig. 1). The leaves and bark of the tree have long history of medicinal uses. It is now widely cultivated, distributed and the fruits enrich the diets of millions of people in the tropics of the world. It is a genus of about 133 genera and more than 3800 species of tropical shrubs and a small tree of 10m height with spreading branches which can survive on all kinds of soils. It is also known as ‘poor man’s apple of the tropics’ has a long history of traditional use [1, 2]. A number of metabolites in good yield and some have been shown to possess useful biological activities belonging mainly to phenolic, flavonoid, carotenoid, terpenoid and triterpene. P. guajava is antispasmodic and antimicrobial properties in the treatment of diarrhoea and dysentery, has also been used extensively as a hypoglycemic agent. Many pharmacological studies have demonstrated the ability of this plant to exhibit antioxidant, hepatoprotection, anti-allergy, antimicrobial, antigenotoxic, antiplasmodial, cytotoxic, antispasmodic, cardio active, anticough, antidiabetic, anti-inflammatory and antinociceptive activities, supporting its traditional uses. Suggest a wide range of clinical applications for the treatment of infantile Rota viral enteritis, diarrhoea and diabetes [3].

Moringa oleifera Lam is the most widely cultivated species in the genius Moringa, the only genus in the plant family Mornigaceae (fig. 2). The common names include Moringa, Drumstick tree, Horse raddish tree and Ben oil tree or Benzoil tree. Moringa has been used from long ago in different parts around the globe as a traditional culture in the herbal medicine reported for effective ailments ranging from inflammations to various gouts and fevers. Moringa leaves rubbed against any temple may relieve severe headaches. There is an anti-inflammatory and anti-bacterial effect when applied to insect bites or wounds. Apply just a poultice of fresh Moringa leaves to stop bleeding from any kinds of shallow cut. Moringa extracts may be used against fungal or bacterial skin problems. Eating food products of Moringa are good for the people who are suffering from malnutrition due to the high fiber and protein content. Moringa leaves used as an antidiabetic agent. Moringa leaves treat bronchitis, fevers, ear and eye infections, and painful mucus membrane inflammation [4, 5].

Diabetes mellitus (DM), commonly referred to as diabetes, is a group of metabolic disorders in which there are high blood sugar levels over a prolonged period. Symptoms of high blood sugar include frequent urination, increased thirst, and increased hunger. If left untreated, diabetes can cause many complications. Acute complications can include diabetic ketoacidosis, hyperosmolar hyperglycemic state, or death. Serious long-term complications include cardiovascular disease, stroke, chronic kidney disease, foot ulcers, and damage to the eyes.

Prevention and treatment involve maintaining a healthy diet, regular physical exercise, a normal body weight, and avoiding use of tobacco. Control of blood pressure and maintaining proper foot care are important for people with the disease. Type 1 DM must be managed with insulin injections. Type 2 DM may be treated with medications with or without insulin. Effective measure in Gestational diabetes usually resolves after the birth of the baby.

The aim of the present study is to compare the anti-diabetic activity of aqueous leaf extracts of Psidium guajava and Moringa oleifera and to investigate the anti-diabetic activity of the combined extract of both the plants. The objective of the study is to perform the Glucose diffusion inhibition method and alpha amylase inhibitory assay of the plants under study individually and as well as a combination of aqueous extracts of both the plants.

Fig. 1: Leaves and fruits of Psidium guajava

Fig. 2: Leaves of Moringa oleifera

MATERIALS AND METHODS

Collection and identification of plant material

The leaves of both plants were collected from the hilly regions of Ernakulum District, and were authenticated by Dr. Sr. Tessy Joseph, Professor, Department of Botany, Nirmala College, and Muvattupuzha. Voucher specimen was deposited in the herbarium of Nirmala College, Muvattupuzha, Kerala, India, https:// www.latlong.net/c/?lat=9.958500andlong=76.627937 with a Voucher no: NCBD 3932.

Preparation of extracts

The mature fresh leaves of Moringa oleifera and Psidium guajava were thoroughly washed with water and allowed to dry under shade. Dried leaves were finely powdered and used for extraction. The plant extract is prepared by decoction. The crude drug is boiled in a specified volume of water for defined time. It is then cooled and strained or filtered. This procedure is suitable for the extracting water soluble, heat stable constituents. The starting ratio crude drug to water is fixed, e. g. 1:4, 1:6. The volume is then brought down to one fourth its original volume by boiling during the extraction procedure. The extract is filtered and used as such or processed further concentrated.

In vitro alpha-amylase inhibition assay

Materials required

Enzyme; (0.5 mg/ml) α-amylase in 0.02 Phosphate Buffer (Type VI B: From porcine pancreas, 500000 U) [15.8 U/mg solid at pH 6.9]-Stored at 2-8 °C, USA (A3176) Substrate: Starch 1%w/v, Sodium dihydrogen orthophosphate (NaH2PO4.2H2O), Disodium hydrogen phosphate (Na2HPO4.2H2O), Indicator: Iodine solution 1% w/v, NaCl solution (0.006 M).

Instruments

UV-Visible Spectrometer (Shimadzu UV-1800), Electronic balance–Citizen CY104.

Procedure

Alpha-amylase activity was carried out by starch-iodine method. The concentrations 25µg/ml, 50µg/ml, 75µg/ml and 100 µg/ml of both plant extract were prepared by dissolving in distilled water. 500 µl of plant extract and 500 µl of 0.02M sodium phosphate buffer (pH 7 with 0.006 M NaCl) containing α-amylase solution (0.5 mg/ml) should be taken in each test tube. It is then incubated at 37 °C for 10 min, 500 µl of starch solution (1%) was added, and the mixture was re-incubated for 1h. Next, 0.1 ml of 1% iodine solution was added, and after adding 5 ml distilled water, the absorbance was taken at 565 nm. Sample, substrate and α-amylase blank determinations were carried out under the same reaction conditions [10-12].

Inhibition of enzyme activity was calculated as

Where, A= absorbance of the sample, B= absorbance of blank (without α-amylase), C= absorbance of control (without sample).

Glucose diffusion inhibitory assay

Materials required: 0.15 M sodium chloride solution, Glucose dialysis membrane.

Instruments: Electronic balance–Citizen CY104, Magnetic stirrer–Rotek UV spectrophotometer (Shimadzu UV 1800)

Procedure: 1.1 ml of extract was placed in a dialysis membrane along with glucose solution (0.22 mmol in 0.15 M sodium chloride). Tied the both ends of membrane with the thread and it was then immersed in water containing a beaker of 40 ml 0.15 M NaCl solution and 10 ml distilled water. The beakers were placed in magnetic stirrer and kept at room temperature. 4.5 ml of solution was pipetted out at 30 min. Interval for 2.5 h from beaker and absorbance of the solution was determined at 248 nm by using UV spectrophotometry. On each withdrawal 5 ml solution from beaker the volume was compensated by replacing it with fresh solution similarly prepared. A controlled determination was also done by taking 1 ml of 0.15M NaCl containing 0.22 mmol glucose and 1 ml distilled water in dialysis membrane. The procedure was repeated for each plant extract and to study combined effect of mixture of extract [13-15].

Statistical analysis: The values obtained are analysed by one-way anova method. Results are expressed as mean±SEM.

RESULTS AND DISCUSSION

The mixture (1:1) of aqueous plant extracts (at a concentration of 100µg/ml) of Psidium guajava linn and Moringa oleifera lam exhibited 72.08333% inhibition with IC50 value of 10.9µg/ml. The leaf extracts of Psidium guajava (at a concentration 100µg/ml) exhibited 71.23288% of a α amylase inhibitory activity with an IC50 values 19.883µg/ml whereas the leaf extracts of Moringa oleifera (at a concentration of 100µg/ml) exhibited 70.58824% of α amylase inhibitory activity with an IC50 value of 27.974 µg/ml. The acarbose (standard drug) at a concentration of 100µg/ml showed 72.09302% inhibitory effect on the α-amylase activity with an IC50 value 8.9µg/ml. The values are tabulated in table 1. The mixture showed more inhibitory effects than the individual plant extracts. By comparing the both plant extracts Psidium guajava leaves showed more inhibitory effect than the leaves of Moringa oleifera.

Table 1: Alpha amylase inhibitory activity of standard drug acarbose, aqueous extract of Psidium guajava (Pg), Moringa oleifera (Mo) and the 1:1 mixture of aqueous extracts of Psidium guajava and Moringa oleifera at 25 µg/ml, 50 µg/ml, 75 µg/ml and 100 µg/ml with respect to control. Results are expressed as mean±SEM. The percentage inhibition was compared with standard acarbose and the IC50 values are 19.883, 27.974 and 10.9 respectively. The mixture has least difference from the standard acarbose, when compared with others.

Table 1: Alpha amylase inhibitory activity of different concentrations of acarbose and the plant extracts on porcine alpha amylase. Results are expressed as mean±SEM.

S. No. Sample Concentration (µg/ml) Absorbance at 565 nm mean±SEM Control mean±SEM Percentage of inhibition IC50
1 Acarbose (Standard) 25 0.172±0.0012 0.048±0.0008 53.21888 8.9
50 0.191±0.0017 0.051±0.0005 60.86957
75 0.216±0.0005 0.057±0.0003 70.98214
100 0.221±0.0006 0.066±0.0014 72.09302
Blank 0.281 - -
2

Aqueous extract of Psidium guajava

(Pg)

 25 0.160±0.0001 0.046±0.00088 49.13793 19.883
50 0.189±0.0012 0.048±0.00033 61.30435
75 0.206±0.0008 0.051±0.00033 68.28194
100 0.215±0.0011 0.059±0.00153 71.23288
Blank 0.278 - -
3

Aqueous extract of Moringa oleifera

(Mo)

 25 0.147±0.00033 0.039±0.000333 49.76959 27.974
50 0.159±0.00115 0.042±0.002309 54.6729
75 0.184±0.00088 0.048±0.000577 65.38462
100 0.196±0.00088 0.0523±0.000667 70.58824
Blank 0.256 - -
4 1:1 mixture of Pg and Mo 25 0.181±0.00577 0.049±0.002028 51.76471 10.9
50 0.211±0.0003 0.053±0.000577 62.94821
75 0.222±0.001528 0.061±0.000333 66.25514
100 0.237±0.00145 0.064±0.001202 72.08333
Blank 0.304 - -

The invitro glucose diffusion inhibition assay of both the plant extracts was carried out to determine the percentage inhibition of glucose diffusion at different time intervals 30, 60, 90, 120 and 150 min. The values are tabulated in table 2. The aqueous extract of Psidium guajava leaves exhibited maximum glucose diffusion inhibition (75.32%) at 150 min as well as Moringa oleifera leaf extract showed the maximum inhibition of 73.70% at the same time interval. By comparing both the plant extracts, Psidium guajava exhibited more inhibitory effects than Moringa oleifera leaves. The mixture of plant extracts exhibited 76.57% inhibition at 150 min which produces more effects than the two plants. The invitro glucose diffusion inhibition assay was found to increase with time from 30 to 150 min and both plants extracts demonstrated significant inhibitory effects on the movement of glucose into external solution across dialysis membrane as compared to control. The retardation of glucose diffusion by Psidium guajava extract was significantly higher than Moringa oleifera. The hypoglycemic effect inhibited by the extract of Psidium guajava and Moringa oleifera is mediated by increasing glucose absorption or by decreasing glucose diffusion rate. At the same time both plant extracts showed significant inhibitory effect produced as compared to standard drug. The combination of the plant extract showed a synergistic effect with higher glucose diffusion inhibitory effect.

Table 2: The invitro glucose diffusion inhibition assay of standard drug acarbose, aqueous extract of Psidium guajava (Pg), Moringa oleifera (Mo) and the 1:1 mixture of aqueous extracts of Psidium guajava and Moringa oleifera at different time intervals 30,60,90,120,and 150 min was performed and percentage of inhibition was calculated with respect to test and control. Results are expressed as mean±SEM. The percentage inhibition was compared with standard acarbose. The mixture has least difference from the standard acarbose, when compared with others.

Table 2: Percentage of inhibition of various plant extracts in Glucose diffusion inhibitory assay at different time intervals using dialysis membrane, results are expressed as mean±SEM,

S. No. Sample Time min Control mean±SEM Test mean±SEM Percentage of inhibition
1 Acarbose (Standard) 30 0.491±0.00057 0.143±0.000577 77.32
60 0.569±0.00057 0.135±0.000453 78.03
90 0.578±0.00175 0.131±0.000333 78.71
120 0.598±0.00057 0.123±0.00033 80.10
150 0.620±0.00023 0.115±0.00088 82.74
2

Aqueous extract of Psidium guajava

(Pg)

 30 0.491±0.00057 0.160±0.00033 67.41
60 0.569±0.00057 0.159±0.00033 72.05
90 0.578±0.00175 0.158±0.00033 72.66
120 0.598±0.00057 0.156±0.000577 73.91
150 0.620±0.00023 0.153±0.00882 75.32
3

Aqueous extract of Moringa oleifera

(Mo)

 30 0.491±0.00057 0.175±0.00033 64.35
60 0.569±0.00057 0.172±0.000577 69.77
90 0.578±0.00175 0.169±0.000333 70.76
120 0.598±0.00057 0.167±0.000333 72.07
150 0.620±0.00023 0.163±0.000333 73.70
4 1:1 mixture of Pg and Mo 30 0.491±0.00057 0.129±0.00045 70.87
60 0.569±0.00057 0.125±0.000667 72.50
90 0.578±0.00175 0.123±0.000333 73.31
120 0.598±0.00057 0.119±0.000667 74.94
150 0.620±0.00023 0.107±0.002028 76.57

Insulin resistance, which is the reduced response of target tissues such as the skeletal muscle, liver, and adipocytes, to insulin, plays a major role in the pathogenesis of Type II Diabetes. Skeletal muscle is the predominant site of insulin-mediated glucose uptake in the postprandial state. Normal glucose homeostasis depends on a well-balanced interaction between tissue (muscle, liver, and fat) sensitivity to insulin and insulin secretion [16]. The present study showed that, the ability of the combined aqueous extract of Psidium guajava and Moringa oleifera to increase the percentage glucose uptake is comparable to that of the standard drug Acarbose, showing its ability to reduce, post prandial blood sugar level.

Type II Diabetes mellitus can be treated by reducing post prandial hyperglycemias. The intestinal digestive enzyme, alpha amylase is a carbohydrate hydrolyzing enzyme. Alpha amylase inhibitors prevent break down of polysaccharide in to mono and disaccharide. Thus Alpha amylase inhibitors can prevent the postprandial hyperglycaemia by preventing glucose release from starch and delaying carbohydrate metabolism [17]. This research demonstrated the, better antidiabetic potential of the 1:1 mixture of aqueous extract of Psidium guajava and Moringa oleifera leaves, by its ability to inhibit Alpha amylase more effectively than either of the individual plant extracts of Psidium guajava and Moringa oleifera leaves.

The results of the Alpha amylase activity and glucose diffusion inhibition assay demonstrated that even though the individual aqueous plant extracts of Psidium guajava and Moringa oleifera, could increase the glucose uptake and inhibit the Alpha amylase activity, the combined aqueous extracts of both the plants increased the glucose uptake and inhibited the alpha amylase more efficiently than individual plants. So the combined aqueous extract (1:1 mixture) of Psidium guajava and Moringa oleifera can act as a better antidiabetic agent. Plants containing various phytochemicals that exhibit additive and synergistic interaction in antidiabetic properties which is a beneficial effect, which may help to formulate more potent antidiabetic drugs in combination [18].

CONCLUSION

The Antidiabetic properties of Psidium guajava and Moringa oleifera was evaluated in vitro by glucose diffusion inhibition assay and alpha amylase inhibition assay. The results of our study indicates that aqueous extract of Psidium guajava and Moringa oleifera inhibit the breakdown of complex carbohydrate as well as reduces the rate of absorption of glucose. Also the potentiation effect shown by the combination of extract may be due to synergistic effect of the phytochemical constituents. Further studies are recommended to find the mechanism behind this synergistic or additive effect. This study also suggests that the active compounds isolated from these plants can be used as lead compound for designing potent anti-diabetic drugs. Based on the future investigations the plants can be utilised as the components of polyherbal formulations for treating diabetes.

ACKNOWLEDGEMENT

The authors are thankful to Nirmala College of Pharmacy Muvattupuzha for providing facilities to perform the studies and also express a sincere gratitude to Dr. Deepa Jose and Elseena Jose of Nirmala College of Pharmacy.

FUNDING

Nils

AUTHOR CONTRIBUTIONS

Mathew Anu Jayamol and Reji Ashly conducted the experiment and prepared the manuscript. Johns Nithya designed the experiment and Shamsudeen Shijina contributed in experimental part of the work.

CONFLIT OF INTERESTS

The authors confirm that this article content has no conflict of interest.

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