Preliminary Phytopharmacological Analysis, Proximate Composition Analysis and Product Development with the Leaves and Fruits of Muntingia calabura L.

 

Angela Elisabeth Peter¹*, Thonta Rama Satyavani², Godavarthi Amenbaby²,

Sai Venkata Kiran Ulabala², Pola Sudhakar¹, Maddali Rajeswari²,

Bhagavathula Venkata Sandeep¹, Battu Ganga Rao³, Veerathu Lakshmi Kalpana¹

¹Department of Biotechnology, College of Science and Technology, Andhra University, Visakhapatnam,

Andhra Pradesh, India.

²Department of Food, Nutrition and Dietetics, College of Science and Technology, Andhra University, Visakhapatnam, Andhra Pradesh, India.

³College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh, India.

 

ABSTRACT:

Muntingia calabura, commonly known as the Jamaican cherry tree is a widely distributed, fast-growing avenue tree that can be easily identified by its green canopy and small, red cherry-like fruits. This study was carried out to evaluate the in vitro antioxidant, antimicrobial and anti-arthritic activity of the chloroform and methanol extract of the leaves and fruits of M. calabura collected from Visakhapatnam, India. The moisture content, ash value, the amount of calcium, vitamin C, fiber and fat, the phosphorous iron, calcium, copper, potassium, magnesium, sodium, zinc and protein content in the leaves and green fruits of M. calabura were determined by standard protocols. Five items (one health drink, three spicy snack items and two sweet snack items) in total were prepared with the selected sample and organoleptic evaluation of the developed products was done by 11 selected panel members. Nutritive values were computed for all the recipes based on the nutrient information database of raw ingredients. The chloroform and methanol extracts were rich in a diverse array of phytochemicals like alkaloids, glycosides, phenolic compounds, tannins, phytosterols, flavonoids and triterpenes. Considerably high free radical scavenging activity (chloroform extract 91.9459±0.25% at a concentration of 100µl and methanol extract 81.0405±10.35% at a concentration of 300µl) and anti-arthritic activity (chloroform extract 96.6983±0.20% at a concentration of 100µl and methanol extract 98.1407±0.05% at a concentration of 600µl) was exhibited by both extracts. While the chloroform extract exhibited greater antifungal activity, the methanol extract showed greater antibacterial activity. Proximate composition analysis revealed that the leaves had almost equal amounts of moisture (54±0.5%) and fiber (54.5%). The green fruits had high moisture content (74.685±1.59%) and a significant amount of crude protein (2.0424±0.01 mg/g). Significant amounts of potassium, phosphorus and iron were found in both the leaves and green fruits of M. calabura. Both the leaves and green fruits had high amounts of magnesium (18.5672±0.6 mg/g and 18.4847±0.23 mg/g respectively) and sodium (13.0325±1.27mg/g and 12.735±1.48mg/g). Calcium and zinc were present in low amounts in the leaves and green fruits. Trace amounts of vitamin C was found in both the leaves (0.0645µg/100g) and the green fruit (0.0166µg/100g) samples of M. calabura. The five food items prepared were well accepted and appreciated for good taste, flavor, appearance and texture. In conclusion, M. calabura is a wonder plant loaded with phytochemicals and minerals having potent pharmacognostic activity and the tea of the leaves and the green/ripe fruits can be innovatively incorporated into food items and consumed to avail the health benefits of this plant.

 

 

INTRODUCTION:

Medicinal plants have been in use for the treatment and cure of various disease conditions. Ancient texts have journalled the use of specific parts of plants for use on the onset of various sicknesses. The WHO report depicts that more than 80% of world’s population rely on plant based products to meet health care needs¹.

 

In living systems, free radicals are generated as part of the body's normal metabolic process². A free radical is any species capable of independent existence that contains one or more unpaired electrons which reacts with another molecule by taking or giving electrons and because of this highly reactive nature, it is involved in many pathological conditions³. Increasing evidence suggests that oxidative stress induced biochemical changes are crucial etiological factors in several chronic human diseases such as diabetes mellitus, cancer, atherosclerosis, arthritis, inflammation and neurodegenerative diseases⁴. Antioxidants are compounds that help to inhibit the many oxidation reactions caused by free radicals thereby preventing or delaying damage to the cells and tissues of living organisms⁵. Recent studies indicate that carotenoids, tocopherols, ascorbates and phenols are strong natural antioxidants which are found in abundance in plants⁶.

 

An anti-microbial is a substance that kills or inhibits the growth of microorganisms⁷. The treatment of infectious diseases with antimicrobial agents continues to present problems in modern day medicine with many studies showing a significant increase in the incidence of side effects and resistance that pathogenic microorganisms build against several antibiotics⁸. Plant derived drugs serve as a prototype to develop more effective and less toxic medicines⁹.

 

Rheumatoid arthritis is an autoimmune disease in which there is joint inflammation, synovial proliferation and destruction of articular cartilage¹⁰. The prevalence of rheumatoid arthritis is around 1% worldwide, with women suffering 3-5 times more than men¹¹. In recent years, use of herbal origin medicines for the treatment arthritis has been gaining momentum¹².

 

Many medicinal plants also happen to be food plants, that are consumed as part of the diet regularly; or that can be eaten occasionally. The Jamaican Cherry Tree (Fig. 1), scientifically known as Muntingia calabura, is a tree that is commonly found in the tropical and sub-tropical regions of the world.

 

It can be found growing wildly or as an avenue tree. The use of various parts of this tree has been documented for medicinal purposes.

 

Figure 1 A- Muntingia calabura canopy-structured tree; B- flower; C- green fruits; D- ripe fruit

 

The roots of M. calabura have been in use in Malaysia as an abortifacient agent and in Vietnam they are used as an emmenogogue¹³. The bark is traditionally used as an antiseptic and for reducing swelling in lower extremities¹⁴. The leaves of M. calabura are known for their anti-nociceptive and pain-relieving activity¹⁵. They are used for the preparation of a ‘tea’ with warm water that can be consumed for pain relief during headaches, cold¹⁶ and gastric ulcers¹⁷. The flowers are used in the treatment of abdominal cramps and spasms of pain¹⁸. The fruits of this tree are edible and are sweet to taste and possess anti-diabetic activity¹⁹. Several studies have been carried out to scientifically validate this traditional knowledge and to isolate the compounds responsible for these medicinal properties.

 

This paper presents the results of the preliminary phytochemical, pharmacological and nutritive study carried out on the leaves and fruits of M. calabura collected from Visakhapatnam district, Andhra Pradesh state in India.

 

MATERIAL AND METHODS:

Plant material:

The leaves and fruits of Muntiagia calabura were collected from Andhra University campus, Visakhapatanam, Andhra Pradesh, India, during the month of January 2017 and January 2018. For all analysis, leaves collected were fresh, green mature leaves (not young and underdeveloped or old). For phytopharmacological analysis, green but fully developed fruits were collected. For most product preparations, green, fully developed fruits were collected and used, but for few (as mentioned appropriately) fully ripe, red fruits were collected.

 

Chemicals:

All chemicals and solvents were of the analytical grade obtained from S.D. Fine Chemical Pvt. Ltd., Mumbai, Sigma Chemical Company, U.S.A., Loba Chemie Pvt. Ltd., Mumbai.

 

Extraction Process:

The fresh leaves and fruits were extracted together, in the two solvents, chloroform and methanol. 30g of fresh leaves and 15 green fruits were extracted together in 40 ml of each solvent. Extraction was done by grinding the fresh sample in ample amount of the chloroform/ methanol using a mortar and pestle. This process facilitates the obtaining of chemical constituents that are soluble in the respective solvents depending upon their solubility and the polarity of the solvents. The two extracts thus obtained were concentrated under vacuum at a temperature of 51°C by using a rotary evaporator²⁰.

 

Preliminary Phytochemical Analysis:

The chloroform and methanol extracts obtained were tested for the presence of phytosterols, triterpenes, glycosides, saponins, phenols, flavonoids, tannins, carbohydrates, alkaloids, quinines and oils by standard qualitative tests²¹.

 

In-vitro Antioxidant Activity:

The free radical scavenging activity of the chloroform and methanol extract of Muntingia calabura were assessed by the DPPH assay method²². An aliquot of 3 ml of 0.004% DPPH solution in Methanol and 0.1ml of plant extract at various concentrations were mixed. The mixture was shaken vigorously and allowed to reach a steady state at room temperature for 30 min. Decolorization of DPPH was determined by measuring the absorbance at 517nm. A control was prepared using 0.1ml of the respective vehicle in the place of plant extract/ascorbic acid. The percentage inhibition activity was calculated as [(A₀-A₁)/A₀] ×100, where A₀ was the absorbance of the control, and A₁ was the absorbance of the plant extract/ ascorbic acid. The IC₅₀ values are computed from the graph plotted with the concentration of the extract on the X-axis and the percent inhibition on the Y-axis.

 

In-vitro Anti-microbial Activity:

In vitro antimicrobial activity of the two extracts of M. calabura was determined by the cup-plate method against selected bacteria and fungi. The cup-plate method is based on measuring the diameter of the zone of inhibition of microbial growth surrounding the cups containing various dilutions of extracts.

 

Antibacterial activity:

The Nutrient agar media was prepared and poured into sterile Petri-plates by pour plate method. After solidification, 20µl of bacteria culture was spread on the agar surface with a sterile L-shape glass rod. Few minutes post inoculation, a sterile metal borer was used to prepare the wells of 4mm diameter in the agar plates. Then the test samples/ plant extracts (50µl, 100µl and 200µl) and the standard (50µl) were added into the wells using a micropipette. All the Nutrient agar plates were incubated at 28ºC for 48-72hr. The presence of a definite zone of inhibition of any size around the well indicated antibacterial activity. The solvent controls were tested simultaneously to assess the activity of DMSO and water which were used as a vehicle. The diameter of the zones was measured and recorded²³. Bacteria employed for the test were obtained from NCL, Pune. Two Gram positive bacteria; Staphylococcus aureus (NCIM No. 2127), Bacillus subtilis (NCIM No. 2063) and two Gram negative bacteria; Pseudomonas aeruginosa (NCIM No. 5029), Klebsiella pneumoniae (NCIM No. 2957) were used. Streptomycin (50µg) was used as positive control.

 

Antifungal activity:

The potato dextrose agar plate surface was inoculated by spreading a volume (20µl) of the fungal inoculum over the entire agar surface. The test samples/ plant extracts (100µl, 300µl and 600µl) were added into wells prepared and the extracts/antifungal agent diffuses in the agar medium and inhibits the growth of the strain tested. Test organisms Aspergillus niger (NCIM No. 839) and Aspergillus flavus (NCIM No. 1028) were used for antifungal assay. An incubation period of 4-5 days was found necessary for culture growth and to read the zone of inhibitions²⁴. Fungi used for the test were obtained from NCL, Pune. Nystatin (50µg) was used as positive control.

 

In vitro Anti-arthritic activity:

Anti- denaturation study is performed by using bovine serum albumin (BSA). When BSA is heated it undergoes denaturation and expresses antigens associated with type- III hypersensitivity reaction and that is related to disease such as serum sickness, glomerulonephritis, rheumatoid arthritis and systemic lupus erythematosus²⁵. The reaction mixture consisted of 0.45ml of 5% bovine serum albumin and 0.05ml of extract of various concentration (100µl, 300µl and 600µl). The product control is prepared with 0.45ml distilled water and 0.05 ml of extract of various concentration (100µl, 300µl and 600µl). The test control which represents 100 % protein denaturation was prepared with 0.45ml of 5% bovine serum albumin and 0.05ml of distilled water. The pH was adjusted to 6.3 using 1N HCl when needed. The samples were incubated at 37°C for 20 minutes and then the temperature was increased to keep the samples at 57° C for 30 minutes. After cooling, 2.5ml of phosphate buffer was added to the above solutions. The absorbance was measured at 660nm²⁶. The percentage inhibition of protein denaturation can be calculated as:

 

Percentage Inhibition = 100 – [{(optical density of test solution– optical density of product control)/optical density of test control} × 100]

 

The IC₅₀ values are computed from the graph plotted with the concentration of extract on the X-axis and the percent inhibition on the Y-axis.

 

Proximate Composition Analysis:

The proximate composition analysis involves the study of different parameters such as the moisture, ash, lipid, protein and other mineral contents in various food samples. This analysis is essential for product development, quality control or regulatory purposes in the food industry. The moisture content and ash value in the leaves and green fruits of M. calabura were determined by the AOAC method²⁷. The amount of calcium, vitamin C, fiber and fat in the leaves and fruits of M. calabura were estimated by protocol described by Raghu Ramulu et al., (1983)²⁸. The phosphorous content in the selected samples was estimated by Fiske and Subbarow method (1925)²⁹. The iron content was estimated by Wong’s method (1928)³⁰. The protein content in M. calabura leaves and fruits was assessed by the protocol described by Lowry et al., (1951)³¹. The calcium, phosphorus, copper, potassium, magnesium, sodium and zinc content in the leaves and green fruits of M. calabura were determined by inductively coupled plasma method using Inductively coupled plasma mass spectrometry (ICP-MS), method APHA, 3120-B³².

 

Product Development:

Five food items were prepared with the incorporation of the leaves and fruits of M. calabura. While the fruits were used directly, the leaves were used to prepare a tea (leaves added to boiling for 2 minutes; water strained and used as tea) that was incorporated into the food items. Jeera water is a health drink made frequently in the kitchens of Indian households that is prepared by boiling jeera (cumin seeds) in water for about 10 minutes. This drink is very good for the stomach, soothes gastritis, helps clearing the gastrointestinal and urinary system and also tones the skin. 200ml of jeera (cumin) water was prepared containing 100ml of tea prepared from the leaves of M. calabura. Three spicy items commonly prepared as tea time snacks in India were also made. Sprout salad; made with raw sprouted green gram, tossed together with finely chopped onion, tomato, capsicum and carrot, chaat; made with chickpeas cooked in a gravy of onion, tomato and spices and chapati; an unleavened thin bread-like pancake made with wheat flour were prepared. Sprout salad and chaat were prepared and garnished with a mixture of lime juice and the tea of leaves of M. calabura. The salad and chaat were topped with raw green fruits of M. calabura. For the preparation of chapatis, the dough was made using equal volumes of water and tea of M. calabura leaves. One sweet snack item; fruit salad was also made. Fruit salad was prepared by tossing together chopped apple, banana, pineapple, grapes, guava and the ripe fruits of M. calabura. It was garnished with almonds and a mixture of honey and tea of M. calabura leaves.

 

Organoleptic evaluation of the developed products:

Sensory evaluation/organoleptic evaluation of the developed products were done by 11 selected panel members. All the products were evaluated on the basis of various parameters like taste, appearance, texture, flavor and overall acceptability³³. Each parameter was evaluated on a five-point scale by various scores, which are as follows:

 

SCORES:

1.     Not acceptable

2.     Average/ satisfactory

3.     Good

4.     Very Good

5.     Excellent

 

Computation of Nutritive value of the developed products:

Nutritive values were computed for all the recipes based on the nutrient information database of raw ingredients³⁴.

 

RESULTS:

Extraction:

The leaves and fruits of M. calabura were together subjected to extraction in two different solvents, chloroform and methanol. 30g of fresh leaves and 15 green fruits were extracted together in each solvent. Extraction with methanol gave more yield of extract, 25 ml as compared to that of the chloroform extract of 15 ml. The obtained extracts were dark green in color and highly concentrated, thick and viscous.

 

Preliminary Phytochemical Analysis:

The preliminary phytochemical analysis revealed the presence of phytosterols, triterpenes, glycosides, phenols, flavonoids, tannins, carbohydrates, alkaloids, quinines and oils in both the chloroform and methanol extracts of M. calabura. Saponins were present in only in the methanol extract but absent in the chloroform extract. The results are represented in the Table 1.

 

Table 1 Preliminary Phytochemical Analysis

Phytochemicals	Muntingia calabura
	Chloroform Extract	Methanol Extract
Phytosterols	+	+
Triterpenes	+	+
Glycosides	+	+
Saponins	-	+
Phenols	+	+
Flavonoids	+	+
Tannins	+	+
Carbohydrates	+	+
Alkaloids	++	++
Quinones	+	+
Oils	+	+

Notes: + = Present, – = Absent, ++= Present, extra prominent coloration observed

 

Antioxidant activity:

Among the two extracts, the chloroform extract showed a higher free radical scavenging activity when compared to the methanol extract. For the chloroform extract, highest percent inhibition was seen at a concentration of 100µl; 91.9459±0.25%, followed by 82.5412±2.30% at a concentration of 300µl and 69.9349±4.05% at an extract concentration of 600µl. The highest activity of the methanol extract was observed at a concentration of 300 µl; 81.0405 ±10.35%, followed by 78.6143±13.38% at a concentration of 100µl and 65.2075±3.12% at an extract concentration of 600µl. The IC₅₀ for the chloroform extract of the leaves and fruit of M. calabura was found to be 82.937µl/ml and that of the methanol extract was 67.730µl/ml. The results are represented in the Fig. 2.

 

Figure 2 Antioxidant Activity of M. calabura Extracts

 

Antimicrobial activity:

The antibacterial activity of the chloroform and methanol extracts of M. calabura was assessed using two Gram-positive and two Gram-negative bacteria. Between the two extracts, methanol extract showed greater antibacterial activity. The results are exhibited in Table 2.

 

Table 2 Antibacterial Activity of M. calabura

Concentrations  (µl)	Extract	Zone of Inhibition (mm)
		S. aureus	B. subtilis	P. aeruginosa	K. pneumoniae
50 µl	C	-	10	-	11
	M	14	16	16	20
100 µl	C	10	10	11	11
	M	18	18	18	12
200 µl	C	11	15	11	12
	M	18	22	19	18
Streptomycin	C	13	24	12	-
	M	16	18	22	21

C- Chloroform extract; M- Methanol extract

 

The antifungal activity of the chloroform and methanol extracts of M. calabura was studied against two commonly observed fungi. The chloroform extract showed greater activity when compared to the methanol extract, but less than the standard antifungal agent, Nystatin. The results are represented in the Table 3.

 

Table 3 Antifungal Activity of M. calabura

Concentrations (µl)		Zone of inhibition in (mm)
		Aspergillus niger	Aspergillus flavus
100 µl	C	10	11
	M	10	-
300 µl	C	10	12
	M	-	-
600 µl	C	11	-
	M	-	-
Nystatin	C	21	-
	M	19	-

C- Chloroform extract; M- Methanol extract

 

Anti-arthritic activity:

The chloroform and methanol extracts were tested for their anti-arthritic potential. All the extracts exhibited good activity, as tested by the inhibition of protein denaturation method. Exceptionally high activity was exhibited by the chloroform extract, 96.6983±0.20% at a concentration of 100µl, 96.148±0.30% at a concentration of 300µl and 95.4977±0.15% at an extract concentration of 600µl. The methanol extract also showed high activity; on par with the chloroform activity at a concentration of 600µl. Percent inhibition at an extract concentration of 100µl for methanol extract was 49.4997±0.77%, for 300µl was 65.1825±17.20% and for 600µl percent inhibition of protein denaturation was 98.1407±0.05%. IC₅₀ values of chloroform and methanol extract are 95.687µl/ml and 1928.182µl/ml respectively Results are expressed in the Fig. 3.

 

Figure 3 Anti-arthritic Activity of M. calabura Extracts

 

Proximate Composition Analysis:

The leaves and green fruits of Muntingia calabura were analysed for fourteen important components, namely moisture content, ash value, phosphorous, fiber content, protein, fat, iron calcium, phosphorus, copper, potassium, magnesium, sodium, zinc and vitamin C content. The fruits of M. calabura had a high moisture content of 74.685±1.59%, which was much higher than that of the leaves, 54±0.5%. The ash value of the leaves of M. calabura was found to be 6.7634±0.21% and that of the fruit was 5.4727±0.77%, as determined by AOAC method. The leaves had a significant fiber content of 54.5%, while the fruits had a relatively low fiber content of 17% only. Both the leaves and fruits had low protein and fat content. Iron content in the leaves was found to be 1.1249±0.04 mg/100g while that in fruits was found to be 2.0833 mg/100g. Very low amounts of calcium were found in the leaves and fruits of M. calabura. The green fruits of M. calabura were rich in phosphorus when compared to the leaves, with the phosphorous content in the fruits being 4.3452±0.05mg/100g and that in the leaves being 1.2640±0.04mg/100g. Both the leaves and green fruits were found to contain significant amount of copper and zinc; 0.1641 mg/g and 0.9067±0.12mg/g and 0.1976mg/g and 0.6332±0.01 mg/g respectively. Both leaves and green fruits were found to contain high amounts of magnesium and sodium. They are also a good source of potassium. Trace amounts of vitamin C only were found in both the leaves and the fruit samples of M. calabura. Results are represented in Table 4.

 

Table 4 Proximate Composition Analysis of M. calabura

Proximate Composition Analysis of Leaves and Green Fruits of M. calabura
S.No.	Component	Leaves	Fruits
1.	Moisture Content (%)	54±0.50	74.6850±1.59
2.	Ash Value (%)	6.7634±0.21	5.4727±0.77
3.	Fibre Content (%)	54.50±0	17.00±0
4.	Protein (mg/g)	0.6078±0.01	2.0424±0.01
5.	Fat (mg/g)	0.2357±0	0.4714±0
6.	Iron (mg/100g)	1.1249±0.04	2.0833±0
7.	Calcium (mg/100g)	0.0429±0	0.0360±0
8.	Phosphorous (mg/100g)	1.2640±0.04	4.3452±0.05
9.	Copper (mg/g)	0.1641±0	0.1976±0
10.	Potassium (mg/g)	3.7932±0.15	2.7747±0.32
11.	Magnesium (mg/g)	18.5672±0.60	18.4847±0.23
12.	Sodium (mg/g)	13.0325±1.27	12.7350±1.48
13.	Zinc (mg/g)	0.9067±0.12	0.6332±0.01
14.	Vitamin C (µg/100g)	0.0645±0	0.0166±0

Notes: Values represented as Mean±Standard Error

 

Product Development and Sensory Evaluation:

The tea of M. calabura leaves and the fruits were employed for the process of product development. Five products, jeera water, fruit salad, sprout salad, chaat and chapati were developed, as represented in Fig. 4. Organoleptic evaluation was carried out. All products were well accepted and appreciated for their delicious taste, distinct flavor, good appearance and nice texture as represented in Fig. 5.

 

Figure 4 Snacks Prepared with the M. calabura (Tea from leaves and fresh fruits)

 

Figure 5 Sensory Evaluation of Snacks Prepared with M. calabura

 

Nutritive Value of Prepared Products:

The nutritive value of the items prepared was calculated and is represented in detail in Table 5. Fruit salad, sprout salad, chaat and chapathi were found to be good sources of energy (391.3 kCal, 192.15 kCal, 179.13 kCal, 148.40 kCal respectively). The protein content in chaat was high, 65.77g. Chaat and sprout salad were a rich source of calcium (341.2mg and 337.9mg respectively). Jeera water proved to be an excellent health drink, with moderate level of energy; 25.3 kCal, significant but not high amount of carbohydrate; 20.0g, low fat content of 0.24g, limited protein content of 0.78g, calcium content of 9.50mg and low iron content of 0.02mg.

 

 

Table 5 Nutritive value of Items Prepared

S. No.	Item Prepared		Energy (k.Cal)	Protein (g)	Carbohydrates (g)	Fat (g)	Calcium (mg)	Iron (mg)
1	Jeera Water	25.30		0.78	20.00	0.24	9.50	0.02
2	Sprout Salad	192.15		9.92	36.26	1.62	337.90	1.20
3	Chaat	179.13		65.77	33.36	2.32	341.20	2.34
4	Chapati	148.40		5.16	34.46	1.09	28.20	0.02
5	Fruit Salad	391.30		1.77	23.77	3.24	29.55	0.80

 

DISCUSSION:

The plant M. calabura has significantly attracted the attention of the scientific community with numerous pharmacological studies undertaken in the last two decades.

 

The preliminary phytochemical analysis of the collectively extracted leaf and fruit extract of M. calabura collected from Visakhapatnam revealed the presence of different phytochemicals when analyzed by standard protocols. Phytosterols, triterpenes, glycosides, phenols, flavonoids, tannins, carbohydrates, alkaloids, quinines and oils in were found to be present in both the chloroform and methanol extracts. This is in line with the findings of other researchers who studied the phytochemicals present in M. calabura. Singh et al. (2017), reported the presence of phlobatannins in the stem, terpenoids in the leaves, alkaloids, terpenoids and steroids in the flowers and reducing sugar, tannins and terpenoids in the fruits³⁵. Vijayan and and Ann (2016) reported the presence flavonoids, tannins, reducing sugars and phenols in the ethanol extract of the fruit; flavonoids and phenols in the methanol extract of the fruits and terpenoids, flavonoids, tannins, reducing sugars and phenols in the water extract of M. calabura leaf. In the present study, saponins were present in only in the methanol extract but absent in the chloroform extract. This finding was contrary to that of Vijayanand and Ann (2016), who reported the presence of saponins, tannins and reducing sugars in the chloroform extract of the fruit of M. calabura³⁶.

 

Substantial antioxidant activity was exhibited by both the chloroform and methanol extracts of M. calabura collected from Visakhapatnam; the IC50 for the chloroform extract was found to be 82.937μl/ml and that of the methanol extract was 67.730μl/ml.. The free radical scavenging ability of the raw fruit extracts were studied by FRAP and CUPRAC methods by Vijayanand and Ann (2016). While the chloroform extract exhibited highest scavenging activity in the FRAP assay (70%), the ethanol extract showed highest activity in the CUPRAC method (55.6%)³⁶. Preethi et al., 2010 studied the antioxidant potential of the fruits of M. calabura from Tamil Nadu, India. All five extracts (petroleum ether, chloroform, ethyl acetate, butanol and methanol) had the capacity to scavenge DPPH radicals, with IC_{50 ­}values ranging from 350.12±0.88 to 90±0.04µg/ml. Highest superoxide scavenging activity was exhibited by the methanol extract, IC₅₀ value- 79.2±0.04µg/ml and the lowest scavenging activity was seen by the chloroform extract, IC₅₀ value- 378µg/ml. Dose-dependent hydroxyl radical scavenging activity was observed for all five extracts, with methanol and butanol extract showing high activity, IC₅₀- 49.98±0.2µg/ml and 52±0.4µg/ml respectively and considerably low activity by the ethyl acetate and chloroform extract, IC₅₀- 198.2±0.02µg/ml and 280.4±0.8µg/ml respectively. The nitric oxide scavenging activity of the methanol extract of M. calabura fruits was the highest, IC_50­- 187±0.04µg/ml compared to the other four extracts, but it was much lower than the standard used, BHT, IC₅₀- 33.5±2.12 µg/ml³⁷. The results of the current study undertaken are in accordance with the above studies, proving that the leaf and fruit extracts of M. calabura have high free radical scavenging activity and are good dietary antioxidants.

 

The antibacterial activity of the leaf and fruit extracts of M. calabura were studied on S. aureus, B. subtilis and P. aeruginosa and K. pneumonia in the present investigation. Among the two extracts, the methanol extract showed much higher activity than the chloroform extract. This is accordance to the findings of Zakaria et al. (2006). They studied the antibacterial activity of the chloroform, methanol and aqueous extract of the leaves of M. calabura against C. diphtheria, S. aureus, B. cerus, P. vulgaris, S. epidermis, K. rhizophila, S. flexneri, E. coli, A. hydrophila and S. typhi by disc-diffusion method. While the chloroform extract possessed very limited ability to inhibit the growth of the bacteria understudy, the methanol and aqueous extracts exhibited a dose-dependent antibacterial activity³⁸. The antibacterial activity of the fruit extracts of M. calabura were tested against S. aureus (isolated from cheese), B. cerus (isolated from rice), P. aeruginosa (isolated from food handlers), E. coli (isolated from residual water) by disc diffusion method by Mogollon et al. (2018). The maximum zone of inhibition was observed for S. aureus 42.±0.12 mm, followed by B. cerus 38.07±0.39 mm, P. aeruginosa 31.05±1.0 mm and E. coli 19.15±0.72³⁹ mm. The phytoconstituents in M. calabura leaves which are responsible for antibacterial activity and cytotoxicity activity were isolated by bioassay-guided screening by Sufian et al. (2013). Three flavones; 5,7-dihydroxy-3,8-dimethoxyflavone (1), 2′,4′-dihydroxychalcone (2) and 5-hydroxy-3,7-dimethoxyflavone (3) and a chalcone; 3,5,7-trihydroxy-8-methoxyflavone (4) were isolated and the structures were elucidated by spectroscopy. Compound 2 exhibited notable antibacterial activity, with MIC value of 50µg/ml and 100µg/ml against MSSA and MRSA respectively⁴⁰. The leaf and fruit extracts of M. calabura were found to have a limited antifungal activity in the current study. Among the two extracts, the chloroform extract was more potent. Ramasamy et al (2014) studied the antifungal activity of the chloroform, petroleum ether and methanolic extract of the leaves of M. calabura against A. solani, Fusarium oxysporum f. sp lycopersici, Pythium sp., Phytophthora sp., Rhizoctonia solani, A. niger and Colletotrichum sp. The chloroform and petroleum ether extracts showed less zone of inhibition compared to the methanol extract, with zones of inhibition of 2.2, 2.0, 2.0, 1.5, 1.2, 1.4 and 1.0 cm against A. solani, Fusarium oxysporum f. sp lycopersici, Pythium sp., Phytophthora sp., Rhizoctonia solani, A. niger and Colletotrichum sp. respectively⁴¹. The root extracts of M. calabura were found to possess antifungal activity against the following fungal pant pathogens, A.solani, F. oxysporum f. sp. lycopersici, Pythium sp., Phytophthora sp., R. solani, A. niger and Colletotrichum in a study undertaken by Ramasamy et al., (2017)⁴². Antifungal metabolite, stigmasterol was isolated from the root of M. calabura by Ramasamy et al., (2014), which gave MIC value of 1mg/ml when tested against A. solani⁴¹.

 

The anti-arthritic activity of the chloroform and methanol leaf and fruit extract of M. calabura was assessed by inhibition of protein denaturation method. While both extracts exhibited good anti-arthritic activity, the activity of the chloroform extract was higher than that of the methanol extract, with IC₅₀ values being 95.687µl/ml and 1928.182µl/ml respectively. This is in agreement with studies undertaken by the scientific community, as discussed subsequently. The anti-rheumatic activity of the ethanol extract of the leaves of M. calabura and its fractions, n-hexane, ethyl acetate and water fractions (each at a concentration of 100 mg/kg) was ascertained in Wistar rats with rheumatoid arthritis induced by intraplantar injection of Complete Freund’s Adjuvant (CFA) into the right paw (Sarimanah et al., 2017). Methylprednisolone was used as standard at a concentration of 0.68mg/kg body weight. Highest inhibition of inflammation was observed in rats treated with standard drug, 20 days after administration of CFA, 52±8.37%. This was followed by the group treated with the ethanolic extract of the leaves and the group treated with n-hexane fraction, 43±13.04%. In case of the group treated with ethyl acetate fraction, the percent inhibition of inflammation was 38±14.40% and that treated with water fraction was 35±12.25%. The concentration of IL-1β in the foot sole membrane was also determined using an ELISA kit. While the concentration of IL-1β in the control group was 5000±0 pg/ml, the least concentration was observed in the group treated with standard; 2261±1720pg/ml, followed by n-hexane fraction-treated group; 2536±1607pg/ml, water fraction-treated group; 2961±63 pg/ml, ethyl acetate fraction- treated group; 3251±3350pg/ml and that of crude ethanol leaf extract-treated group; 3951±37 pg/ml. This decrease in the IL-1β levels is indicative that the phytoconstituents in M. calabura leaves have a significant effect on the mediators of inflammation⁴³.

 

Proximate composition analysis revealed substantial moisture content, fiber content, copper and zinc levels in the leaves of M. calabura. High amount of iron, phosphorous and potassium and an even higher amount of sodium and magnesium were observed. Protein content and fat content was low in the leaves. Minute quantity of vitamin C is present in the leaves of M. calabura. The moisture content, in the green fruits of M. calabura was found to be significantly high. Fiber content on the other hand is low. Significant amounts of protein, iron, potassium and phosphorus were observed. Low fat content and low levels of zinc and copper were noted. Calcium content is very low. Sodium and magnesium levels are significantly high. The green fruits were also found to have Vitamin C content, lower than that of the leaves. Gayatri et al., (2006) studied the proximate analysis of ripe fruits of M. calabura and reported 78% moisture content, 5.67% total ash content, 0.972% crude fiber content, 87 mg/g total sugar, 10 mg/g crude protein, 17.5mg/g fat content. The presence of anti-nutritive elements like tannins (390-97.2µg/ 100 g sample), phytic acid (6.25-2.5µg/100g sample) and a total phenol content of 8.88-3.93mg/g was reported. Mature fruits were found to be a good source of minerals. Substantial quantities of β-carotene; 113.35 µg/g, 588.23µg/g ascorbic acid and 82.67µg/g tissue tocopherol were observed in mature fruits⁴⁴. The fruits of M. calabura were studied in detail by Karthyaini and Suresh, (2012). A study of the physio-chemical parameters revealed total ash value to be 8.5% w/w, water-insoluble ash contet to be 2.8% w/w, acid insoluble value 3.6% w/w, sulphated ash value 2.3% w/w, alcohol soluble extractive to be 2.5% w/w, water-soluble extractive was established as 2.8 and the loss on drying was determined to be 9.2% w/w⁴⁵. Muslimin et al., (2019) reported 77.26% of moisture, 1.66% ash, 1.63% protein and 0.11% vitamin C in the fruits of M. calabura. They also reported the mineral content in the fruits of M. calabura ; 0.904mg of copper, 5.39mg of iron, 88.22mg of calcium, 1966.8mg of potassium, 66.11 mg of magnesium, 24.948mg of sodium and 110.726mg of phosphorous (mineral content in M. calabura equivalent to 1 cup; 220ml of M. calabura juice)⁴⁶.

 

CONCLUSION:

Muntingia calabura, also known as West Indian cherry is an avenue tree found in abundance in Visakhapatnam, a coastal city in the state of Andhra Pradesh. The different parts of this plant are known to have diverse medicinal properties. The leaves and green fruits of M. calabura were collected and extracted together in two solvents, chloroform and methanol. The obtained extracts were viscous and dark green in color. The presence of phytosterols, triterpenes, glycosides, phenols, flavonoids, tannins, carbohydrates, alkaloids, quinines and oils could be confirmed in the chloroform and methanol extract by the preliminary phytochemical analysis. The chloroform extract showed a higher antioxidant and anti-arthritic activity when compared to the methanol extract. Between the two extracts, the methanol extract showed greater antibacterial activity and the chloroform extract showed greater antifungal activity. The presence of fourteen important components, namely moisture content, ash value, phosphorous, fiber content, protein, fat, iron calcium, phosphorus, copper, potassium, magnesium, sodium, zinc and vitamin C content in the leaves and green fruits of M. calabura were quantitatively analyzed. The leaves and fruits of M. calabura had significant moisture content, the fruits having greater content than the leaves. The leaves had significant fiber content. Both the leaves and fruits had low protein and fat content. High amounts of magnesium and sodium was recorded in both leaves and green fruits. Trace amounts of vitamin C was found in both the leaves and the fruit samples.

 

An attempt was made to prepare five food items by incorporating a tea prepared from the leaves of M. calabura and freshly picked M. calabura fruits. All the five products, jeera water, sprout salad, chaat, chapathi and fruit salad were well accepted and appreciated by the panel of 11 members as assessed by scores given on a five point scale. Fruit salad, sprout salad, chaat and chapathi were found to be good sources of energy. Jeera water proved to be an excellent health drink, with moderate level of energy, low fat content and a significant but not high amount of carbohydrate.

 

In conclusion, M. calabura is a very versatile plant, having a diverse spectrum of phytochemicals and being rich in several vital minerals. This unique combination makes M. calabura an exceedingly important plant for its good pharmacognostic and nutritive value.

 

ACKNOWLEDGEMENT:

The authors wish to thank SV Enviro Labs and Consultants, Visakhapatnam for assistance provided in ICP-MS analysis. The authors also wish to thank Andhra University for the facilities provided.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1.      Tiwari P. Antimicrobial Activity of Drakshasava Prepared by Traditional and Modern Methods. Research Journal of Pharmacognosy and Phytochemistry. 2014; 6(3): 126-128.

2.      Rajendran R, Hemachander R, Ezhilarasan T, Keerthana C, Saroja DL, Saichand KV and Abdullah MG. Phytochemical Analysis and In vitro Antioxidant Activity of Mimosa pudica Lin., Leaves. Research Journal of Pharmacy and Technology.2010; 3(2): 551-555.

3.      Pal R, Girhepunje K, Shrivastava N, Hussain MM and Thirumoorthy N. Antioxidant and Free Radical Scavenging Activity of Ethanol Extract of Morinda citrifolia. Research Journal of Pharmacy and Technology. 2011; 4(8): 1224-1226.

4.      Soni H, Nayak G, Mishra KSinghai AK and Pathak AK. Evaluation of Phyto Pharmaceutical and Antioxidant Potential of Methanolic Extract of Peel of Pucina granatum. Research journal of Pharmacy and Technology. 2010; 3(4): 1170-1174.

5.      Byahatti VV, Khan AM, Patil SH and Choudhury A. Preliminary Phytochemical Investigation and In vitro Antioxidant Activity of Bergenia ciliata Leaves. Research journal of Pharmacy and Technology. 2010; 3(2): 399-401.

6.      Vaghela JS and Sisodia SS. In vitro Antioxidant Activity of Terminalia chebula Fruit Extracts. Research Journal of Pharmacy and Technology. 2011; 4(12): 1835-1843.

7.      Jayasri R and Anuradha R. In vitro Antioxidant and Antibacterial Activity of Boerhaavia diffusa. Research Journal of Pharmacognosy and Phytochemistry. 2012; 4(4): 223-225.

8.      Aleem A, Anjum NF and Kavitha AN. Comparative In vitro Antimicrobial Activity of Three Different Macro Fungus Extracts Against Clinical Isolates of Gram-Negative Pathogens. Research Journal of Pharmacognosy and Phytochemistry. 2012; 4(1): 29-32.

9.      Rastogi T, Ghorpade DS, Deokate UA and Khadabadi SS. Studies on Antimicrobial Activity of Boswellia serrata, Moringa oleifera and Vitex negundo: A Comparasin. Research Journal of Pharmacognosy and Phytochemistry. 2009; 1(1): 75-77.

10.   Singh M, Soni P, Upmanyu N and Shivhare Y. In vitro Anti-arthritic Activity of Manilkara zapota Linn. Asian Journal of Pharmacy and Technology. 2011; 1(4): 123-124.

11.   Sivakumar G, Sivakumar GA and Rebecca. Evalution of Anti-arthritic Activity of Methanolic Extract of Barleria prionitis on CFA Induced Rats. Asian Journal of Pharmacy and Technology. 2019; 9(3): 159-164.

12.   Kumar SA, Venkatarathanamma V, Saibabu NV, Tentu NR, Kota PV, Kumar KS and Vijayalakshmi A. Anti-arthritic Activity of Annona squamosa Leaves Methanolic Extract on Adjuvant Induced Arthritis in Rats. 2017; 9(2): 46-56.

13.   Rajesh R, Jaivel N and Marimuthu P. Antifungal Metabolite from Muntingia calabura Root against Early Leaf Blight of Tomato. Journal of medicinal Plant Research. 2014; 8(17): 646-656.

14.   Mahmood ND, Nasir NLM, Rofiee MS, Tohid SFM, Ching SM, Leh LK, Salleh MZ and Zakaria ZA. Muntingia calabura: A Review on its Traditional Uses, Chemical Properties and Pharmacological Observations. Pharmaceutical Biology. 2014; 52(12): 1598-1623.

15.   Sani MHM, Zakaria ZA, Balan T, Teh LK and Salleh MZ. Antinociceptive Activity of Methanol extract of Muntingia calabura Leaves and the Mechanism of Action Involved. Evidence-Based Contemporary and Alternative Medicine. 2012; 2012: 1-10.

16.   Sarojini S and Mounika B. Muntingia calabura (Jamaican Cherry): An Overview. Pharma Tutor. 2018; 6(11): 1-9.

17.   Balan T, Sani MHM, Suppaiah V, Mohtarrudin N, Suhaili Z, Ahmad Z and Zakaria ZA. Antiulcer Activity of Muntingia calabura Leaves involves the Modulation of Endogenous Nitric Oxide and Nonprotein Sulfhydryl Compounds. Pharmaceutical Biology. 2014; 52(4): 410-418.

18.   Preethi K, Premasudha P and Keerthana K. Anti-inflammatory Activity of Muntingia calabura fruits. Pharmacognosy Journal. 2012; 40(30): 51-60.

19.   Pramono VJ and Santosa R. Effect of Keren Fruit Extract (Muntingia calabura) on Blood Glucose Levels of Rats (Rattus novergicus) which Induced by Streptozotocin (STZ). Jurnal Sain Veteriner. 2014; 32(2): 218-223.

20.   Azwanida NN. A Review on the Extraction Methods Use in Medicinal Plants, Principle, Strength and Limitation. Open Access Journal of Medicinal and Aromatic Plants. 2015; 4(3): 1-6.

21.   Sahira BK and Cathrine L. General Techniques Involved in Phytochemical Analysis. International Journal of Advanced Research in Chemical Science. 2015; 2(4): 25-32.

22.   Braca A, Fico G, Morelli I, Simone D, Tome F and Tommasi N. Antioxidant and Free Radical Scavenging Activity of Flavonol Glycosides from Different Aconitum species. Journal of Ethnopharmacology. 2003; 86(1): 63 – 67.

23.   Cleidson V, Simone MS, Elza FAS and Artur SJ. Screening Methods to Determine Antibacterial Activity of Natural Products. Brazilian Journal of Microbiology. 2007; 38(2): 369-80.

24.   Hadecek F and Greger H. Testing of Antifungal on Natural Products: Methodologies, Comparability of Results and Assay Choice. Phytochemical Analysis. 2000; 11(3): 137-41.

25.   Agrawal SS and Paridhavi M. Herbal Drug Technology. 2nd ed. Hyderabad: University press Pvt. Ltd., 2007.

26.   Alamgeer, Uttra AM and Hasan UH. Anti-arthritic Activity of Aqueous-Methanolic Extract and various Fractions of Berberis orthobotrys Bien ex Aitch. BMC Complementary and Alternative Medicine. 2017; 17: 371-386.

27.   AOAC. Official Methods of Analysis. 16th ed. Arlington, VA: Association of Official Analytical Chemists; 1995.

28.   Raghuramulu N, Nair MK and Kalyan SS. A Manual of laboratory Techniques. 1st ed. Hyderabad, India: National Institute of Nutrition-ICMR; 1983: 178-179.

29.   Fiske CH and Subbarow Y. The Colorimetric Determination of Phosphorous. Journal of Biological Chemistry. 1925; 16(2): 375-400.

30.   Wong SY. Colorimetric Determination of Iron of Iron and Hemoglobin in Blood II. Journal of Biological Chemistry. 1928; 77: 409-412.

31.   Lowry OH, Rosebrough NJ, Farr AL and Randall RJ. Protein Measurement with the Folin Phenol Reagent. Journal of Biological Chemistry. 1951; 193(1): 265–275.

32.   Greenberg AE, Clesceri LS and Eaton AD. Standard Methods for the Examination of Water and Wastewater. 18th ed. Washington DC, USA: American Public Health Association; 1992.

33.   Frank K, Bob FF and Eugene H. Fruit Characteristics and Sensory Attributes of an Ideal Sweet Cherry. Hort Science. 1996; 31(3): 443-46.

34.   Gopalan C, Rama SBV and Balasubramanian SC. Nutritive Value of Indian Foods. 2nd ed. Hyderabad: National Institute of Nutrition; 2011.

35.   Singh R, Iye S, Prasad S, Deshmukh M, Gupta U, Zanje A, Patil S and Joshi S.. Phytochemical Analysis of Muntingia calabura Extracts Possessing Antimicrobial and Anti-fouling Activity. Internaional Journal of Pharmacognosy and Phyochemical Research. 2017; 9(6); 826-832.

36.   Vijayanand S and Ann ST. Screening of Michelia champacca and Muntingia calabura Extracts for Potential Bioactivities. International Journal of Pharma Sciences and Research. 2016; 7(6); 266-273.

37.   Preethi K, Vijayalakshmi N, Shamna R and Sasikumar JM. In vitro Antioxidant Activity of Extracts from Fruits of Muntingia calabura Linn. from India. Pharmacognosy Journal. 2010; 2(14): 11-18.

38.   Zakaria ZA, Fatimah CA, Jais AMM, Zaiton H, Henie EFP, Sulaiman MR, Somchit MN, Thenamutha M and Kasthuri D. The In-vitro Antibacterial Activity of Muntingia calabura Extracts. International Journal of Pharmacology. 2006; 2(4): 439-442.

39.   Mogollon OFC, Gonzalez-Cuello RE and Lopez JSG. In vitro Antibacterial and Antioxidant of Munitingia calabura Fruits Extract. Contemporary Engineering Sciences. 2018; 11(18): 881-890.

40.   Sufian AS, Ramasamy K, Ahmat N, Zakaria ZA and Yousif MIM. Isolation and Identification of Antibacterial and Cytotoxic Compounds from the Leaves of Muntingia calabura L. journal of Ethnopharmacology. 2013; 146(1); 198-204.

41.   Ramasamy R, Jaivel N and Marinuthu P. Antifungal Metabolite from M. calabura Root against Early Leaf Blight of Tomato. Journal of Medicinal Plants Research. 2014; 8(17): 646-656.

42.   Ramasamy R, Nanjundan J and Ponnusamy. Solvent Extraction and Evaluation of Antifungal Activity of Muntingia calabura Root against Fungal Pathogens. International Journal of Current Microbiology and Applied Sciences. 2017; 6(7): 77-83.

43.   Sarimanah J, Adnyana IK, Sukandar EY and Kurniati NF. The Antirheumatic Activity of Muntingia calabura L. Leaves Ethanol Extract and its Fracion. Asian Journal of Pharmaceutical and Clinical Research. 2017; 10(1); 84-86.

44.   Gayatri T, Renjima V, Murugan K and Mohan TCK. Change in the Nutritional, Antinutritional and Antioxidant Characteristics during Development and Ripening of Jamaican Cherry (Muntingia calabura L.). Journal of Phytological Research. 2006; 19(2): 157-164.

45.   Karthyaini and Suresh K, 2012. Pharmacognostic Evaluation, In vitro Antioxidant and In vivo Anti-inflammatory Studies of Muntingia calabura Linn. Journal of Global Trends in Pharmaceutical Sciences. 2010; 3(3): 805-811.

46.   Muslimin L, Rini, Hasyim I, Yusuf NF, Mubarak F and Yulianty R. Nutrient Content, Mineral Content and Antioxidant activity of Muntingia calabura Linn. Pakistan Journal of Nutrition. 2019; 18(8): 726-732.

 

 

Received on 21.11.2019           Modified on 06.01.2020

Accepted on 18.02.2020         © RJPT All right reserved