Screening of Anti-inflammatory and Antimicrobial activity on Flower Extracts of Morinda coreia Buch Ham.

Bharadhan Bose^1,2*, Sethuramani Amarnathan², Gopi Selvam²

¹Sree Abirami College of Pharmacy, (Affiliated with The Tamil Nadu Dr. M. G. R. Medical University, Chennai) Coimbatore - 641021, Tamil Nadu, India.

²College of Pharmacy, Madurai Medical College, (Affiliated with The Tamil Nadu Dr. M. G. R. Medical University, Chennai) Madurai - 625020, Tamil Nadu, India.

*Corresponding Author E-mail: b.barani143@gmail.com

ABSTRACT:

Objectives: Morinda coreia has rich active components which has been used in folk medicine for the treatment of several diseases. Their active substance content may vary according to plant genetics, parts used,as well as to climatic factors, quality of the ground in which the plants were grown, the time of harvesting, and the extraction methods. Basically the flowers can heal the common cold, improve memory, aid relaxation due to presence of volatile oil. Flowers could be used as a new approach for the development of nutraceutical products or functional foods. The flower part of this plant are not documented. To develop and apply it, we report Pharmacological effects such as anti-inflammatory, antimicrobial activity. Methods: The extracts were prepared by cold maceration process with aqueous and organic solvents such as ethanol and ethyl acetate. The anti-inflammatory activity were evaluated using carrageenan-induced paw edema method and the antimicrobial activity by agar well diffusion method. Results: The maximum zone of inhibition for Bacillus subtilis was found to be more susceptible toward the ethanolic extract (14mm) at the maximum conc. of 300µl followed by ethyl acetate (10mm) and aqueous extract (12mm). Similarly for Klebsiella pneumonia maximum inhibitory zone (13mm) followed Pseudomonas auroginosa (13mm), Staphylococcus aureus (12mm), Candida albicans was moderate sensitive towards ethanolic extract (11mm) Conclusion: The ethanolic extract of Morinda coreia flower possessed high anti-inflammatory, antimicrobial activity than ethyl acetate and aqueous extracts on comparing with standards.

KEYWORDS: Morinda coreia, flower, Anti-inflammatory, Antimicrobial activity, Bacterial resistance.

1. INTRODUCTION:

Medicinal plants contain different pharmacologically active compounds that act individually to improve health. Since ancient times, many herbs have been potentially used as an alternative remedies for treatment of many infectious diseases.

The tribal people cure their ailments by using crude drugs and different parts of plants which are locally available. The traditional systems of medicine are still considered as a great knowledge based in herbal medicines¹. For a long period of time, plants have been a valuable source of natural products for maintaining human health. Traditional medicinal plants with rich phytochemical components are commonly used to obtain preparations beneficial for significant wound healing purposes comprising a comprehensive area of various skin-related diseases². The role of medicinal plants and their wound healing activity was observed in numerous studies^3-7. Morinda coreia (Family: Rubiaceae) which forms the subject of this study is traditionally used in Ayurveda and Siddha systems for their phytochemical profile and therapeutic potential. It is commonly called as Indian mulberry or aal or nuna in India. It is a small tree with distinguished medicinal properties. Literature survey reveals that traditionally the leaf juice is given orally before food for easy digestion⁸. The expressed juice of leaves is externally applied to gout to relieve pain. The leaves are administered internally as a tonic and febrifuge⁹. The n-hexane, dichloromethane, and methanol extracts of the leaves were shown to possess anti-bacterial and antifungal activities¹⁰. The petroleum ether extract showed anti-convulsant activity¹¹. Anti-convulsant, analgesic, anti-inflammatory, cytoprotective effect, and anti-microbial activity of M. coreia leaves have been reported¹². Early studies reported that the extract of leaf, root, and fruits of this plant showed anti-bacterial, analgesic, anti-oxidant, anti-inflammatory, astringent, laxative, sedative, and hypotensive (lowers blood pressure) potentials^13-16. The extracts of fruits are commercially available in the form of juice which claimed to reduce hypertension, painful menstruation, arthritis, gastric ulcers, diabetes, and depression.

Morinda coreia leaves ofhas been reported to have anti-convulsant¹⁷, analgesic, anti- inflammatory¹⁸, antioxidant and cytoprotective effect¹⁹. Unripe fruit is used to cure rheumatism. Ash of the fruit prevents dysentery, vomiting, diarrhoea and cholera. There is greater demand for fruit extract of Morinda species in the treatment of arthritis, cancer, gastric ulcer and other heart disease²⁰. The major phyto constituents present in various parts of Morinda coreia are Ursolic acid, Quercetin, Kaempferol-3-rutinoside, Acacetine-7-glucopyranoside, Alizarin-1-Methyl ether, Rubiadin, Morindonin, Damnacanthal, Nordamnacanthal.Antioxidants protects the cell against the damaging effects of reactive oxygen species (ROS)²¹. Oxidative stress has been implicated in the pathology of many diseases such as inflammatory conditions²² diabetes, cardiovascular diseases, cancer, neurodegenerative diseases and aging²³. The plants contain a wide variety of free radical scavenging molecules such as flavonoids, phenols, terpenoid, and vitamins. The plants have long history in the treatment of different cancer cells²⁴. Fruit and leaf of Nuna used in treating arthritis, diabetes, inflammation and pain²⁵. Nuna blossoms applied topically to treat conjunctivitis and red eyes. Nuna blossom eaten to treat kidney and bladder disorder²⁶.

Inflammation is one of the results of free radical damage. When free radical damage is not controlled, chronic inflammation occurs. Chronic inflammation is one of the main ways for development of degenerative diseases. Degenerative diseases are heart disease, diabetes, cancer, arthritis, Alzheimer's, etc. Anti-inflammatory refers to the property of a substance or treatment that reduces inflammation or swelling. Anti-inflammatory drugs make up about half of analgesics, remedying pain by reducing inflammation as opposed to opioids, which affect the central nervous system^27-31.

Based on the literature survey, there is no extensive activity reported on flowers of Morinda coreia. So the current study is undertaken to evaluate the pharmacological screening for extracts of Morinda coreia flower.

2. MATERIAL AND METHODS:

2.1 Materials:

Reagents and solvents used:

Pet Ether, Ethanol, Ethyl acetate, Chloroform, Benzene, Toluene, Formic acid, 0.1M Phosphate buffer, Sulphuric acid, Sodium Phosphate, Carrageenan, Normal saline, Diclofenac sodium, Nutrient broth, Rose Bengal broth, Muller Hinton agar medium, Potato dextrose, Amikacin and Ketaconazlewere purchased from Sigma Aldridge, Mumbai.

Test organisms used:

The microbial cultures Gram positive organism such as Staphylococcus aureus (MTCC 3160), Bacillus cereus (MTCC 9786), Klebsiella pneumoniae MTCC-432 gram negative organisms such as Pseudomonas aeroginosa (MTCC 4673) and fungi such as Candida albicans (MTCC 1637), were used for this study. All the bacterial cultures were obtained from Microbial Type Culture Collection and Gene Bank (MTCC), Sector 39-A, Chandigarh.

Instrument used:

Hot air oven, Incubator, Digital Plethysmometer

2.2 Plant collection:

Fresh plant material of Morinda coreia flower was collected from Virudhunagar District, Tamilnadu, India during September. The plant was identified and authenticated by Botanist Dr. Stephen, Senior Lecturer, American College of Arts and Science, Madurai.

2.3 Extraction:

The powdered Morinda coreia flower was passed through sieve no 40. The coarse powder was stored in an air tight container. The extraction was carried out by cold maceration process, in which 250gm of coarse powder was defatted with 500ml of petroleum ether for 4 hours. Then it was filtered and the residue was air dried. To the air dried residue, 500ml of ethanol, was added and kept aside for 72hours with intermittent shaking. After 72hours the product was filtered and the filtrate was concentrated and dried at room temperature using rotovapour. The same process was carried out to the marc left after filtration to get ethyl acetate and aqueous extracts.

2.4 Anti-inflammatory Activity:

The anti-inflammatory activity was evaluated using carrageenan-induced paw edema method³¹. The inflammation was readily produced in the form of edema with the help of irritant such as carrageenan. Carrageenan is a sulphated polysaccharide obtained from sea weed (Rhodophyceae) and when injected cause the release of prostaglandins by the way it produces inflammation and edema.

The animals were divided into 5 groups each having six animals.

Group-1 Treated as normal - received 10ml/kg of normal saline administered orally.

Group-2 Treated as standard-received 100mg/kg of Diclofenac sodium administered intraperitonealy.

Group-3 Treated with 100mg/kg of Ethyl acetate flower extract of Morinda coreiadissolved in 2ml sterile water and administered orally.

Group-4 Treated with 100mg/kg of Ethanolic flower extract of Morinda coreiadissolved in 2ml sterile waterand administered orally.

Group-5 Treated with 100mg/kg of aqueous flower extract of Morinda coreiadissolved in 2ml sterile water and administered orally.

Procedure:

A freshly prepared suspension of carrageenan (1% w/v, 0.1ml) was injected to the planter region of left hind paw of each rat. One group was kept as control and the animals of the other groups were pre-treated with the various flower extract of Morinda coreiagiven through orally 60min before the carrageenan treatment. The paw volumes of the test compounds, standard and control groups were measured at 60, 240, 360 minutes of carrageenan treatment with the help of Digital plethysmometer (Ugobasile, Italy). Mean increase in paw volume was measured and the percentage of inhibition was calculated and the results are given

Vc - Vt

% Anti-inflammatory activity = ---------------- x 100

Where,

Vt - mean increase in paw volume in rats treated with test compounds,

Vc - mean increase in paw volume in control group of rats.

Statistics:

Data are expressed as Mean±SEM; data analyzed by one way ANOVA followed by Newman’s keul’s multiple range tests to determine the significance of the difference between the control group and rats treated with the test compounds.

* Values were considered significant at P<0.01.

2.7 Anti-microbial Activity:

The agar well diffusion method was employed for the determination of antimicrobial activity³².

Preparation of inoculum:

The bacterial cultures were inoculated into nutrient broth and incubated for 24hour at 37°C. Fungal cultures were inoculated into rose Bengal broth and incubated for 48 hour at 37°C. The turbidity of the medium indicates the growth of organisms.

About 20ml of Muller Hinton agar medium for bacteria and potato dextrose agar for fungus was poured into sterilized Petri dishes and allowed to solidify. The agar medium was spread with 24hrs cultured 108 CFU/ml of microbial strains by a sterilized rod. The wells (6mm in diameter) were made in the agar plates using a sterilized cork borer. About 50,100, 200, 300μl of the extracts (1mg/ml) were poured into the well using a sterile micropipette. Two common antibiotics Amikacin and Ketaconazole (30µgm/ml) were used as reference drug, and corresponding solvents (ethanol, ethyl acetate, water) were used as positive control for the assay. Then the plates were incubated at 37±2°C for 24hours for bacterial activity and 48 hours for fungal activity. The plates were observed for the zone formation around the wells. The zone of inhibition was calculated by measuring the diameter of the inhibition zone around the well (in mm) including the well diameter. The results were tabulated.

3. RESULTS AND DISCUSSIONS:

3.1 - Anti-inflammatory activity:

In carrageenan induced paw oedema study of different extracts (EEMC, EAEMC, AEMC) of Morinda coreia at a dose of 100mg/kg showed 70, 69 and 62% inhibition of paw edemawhile standard drug Diclofenac at 100 mg/kg showed inhibition of paw edema volume at 71% respectively at the end of 6hrs. From the Table 1, the results revealed that the extracts of Morinda coreia (EEMC, EAEMC and AEMC) at a dose of 100mg/kg possesses significant Anti- inflammatory activity when compared to standard. The EEMC possesses most significant Anti- inflammatory activity than EAEMC and AEMC.

Table 2: Anti-microbial activity screening zone of inhibition (in mm)

Conc(µl)	*Bacillus subtilis*			*Klebsiellapnemoniae*			*Pseudomonasauroginosa*			*Staphylococcus aureus*			*Candida albicans*
Conc(µl)	EA	EE	AE	EA	EE	AE	EA	EE	AE	EA	EE	AE	EA	EE	AE
50	4	8	5	5	9	R	6	9	5	6	8	4	R	7	R
100	7	11	7	7	10	4	8	11	7	9	9	6	4	9	3
200	8	12	9	9	11	6	9	12	8	10	11	9	6	10	6
300	10	14	12	11	13	9	10	14	12	11	12	11	7	11	9
Control	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R
Standard Disc	16			17			16			15			14

Fig 1: Antimicrobial activity zone of inhibition images for flower extracts of Morinda coreia

4. CONCLUSION:

The Morinda coreia flower extracts were prepared bycold maceration process. Pharmacological evaluation deals with the comparison of EEMC, EAEMC and AEMC for its in-vivo anti-inflammatory, and in-vitroantimicrobial activities. The in-vivoanti-inflammatory activity for various extracts of Morinda coreia flower was screened by carrageenan induced paw edema method in rats. The results revealed that the extracts of Morinda coreia (EEMC, EAEMC and AEMC) at a dose of 100mg/kg possesses significant Anti- inflammatory activity when compared to standard. The EEMC possesses most significant Anti-inflammatory activity than ethyl acetate and aqueous extract of Morinda coreia flower. The invitro antimicrobial activity was screened by agar well diffusion method against selected micro-organism. This study suggested that, ethanolic extract of Morinda coreia flower has significant antimicrobial activity, while aqueous and ethyl acetate extracts of Morinda coreia has moderate activity against tested micro-organisms

In an attempt to find natural environmental benign, easily available plant based, we have demonstrated the efficiency of Morinda coreia flower extract possessing a variety of fascinating morphologies owing to its diverse groups of phytochemicals like phenols, flavonoids, sterols, tannins and saponins. The important outcome of this study will be the development value added product from medicinal plant Morinda coreia for biomedical and herbal based industries.

5. ACKNOWLEDGEMENTS:

We acknowledge our sincere gratitude and thanks to College of Pharmacy, Madurai Medical College, Madurai, Tamil Nadu for support and providing the lab facilities to carry out this research work in a successful manner.

6. ETHICS DECLARATIONS:

Ethical Approval:

For anti-inflammatory activity screening this work has got an animal ethical clearance permission to use Wistar rats from IAEC KM College of Pharmacy, Madurai, Tamil Nadu. Proposal Number– TNMGRMU/261420707/IAEC/KMCP/205/2015-16. Given Consent to Participate and consent to publish this research work for publication.

7. CONFLICTS OF INTEREST:

The authors declare that there is no conflict of interests regarding the publication of this article.

8. AUTHORS CONTRIBUTIONS:

The corresponding author Bharadhan Bose executed the experimental research work, data collection, processing, analysis, validation, interpretation, and wrote the original manuscript.

Gopi Selvam - Contributed to the experimentation, reviewed, edited, and scientifically modified the manuscript.

Sethuramani Amarnathan - Contributed to supervision, interpretation of data and concluding the results, technical support, manuscript review, and editing functions.

9. REFERENCES:

1. Diet, Nutrition and the Prevention of Chronic Diseases. World Health Organization, Technical Report Series, WHO, Geneva. 2003; 916.

2. Maver T, Maver U, StanaKleinschek K, Smrke DM, Kreft S. A review of herbal medicines in wound healing. Int J Dermatol. 2015; 54(7):740–751. doi: 10.1111/ijd.12766.

3. Elzayat EM, Auda SH, Alanazi FK, Al-Agamy MH. Evaluation of wound healing activity of henna, pomegranate and myrrh herbal ointment blend. Saudi Pharm J. 2018

4. Dons T, Soosairaj S. Evaluation of wound healing effect of herbal lotion in albino rats and its antibacterial activities. Clin Phytosci. 2018; 4(1): 6. doi: 10.1186/s40816-018-0065-z.

5. Pereira LOM, Vilegas W, Tangerina MMP, Arunachalam K, Balogun SO, Orlandi-Mattos PE, de Oliveira Martins DT. Lafoensiapacari A. St.-Hil.: wound healing activity and mechanism of action of standardized hydroethanolic leaves extract. J Ethnopharmacol. 2018; 219:337–350. doi: 10.1016/j.jep.2018.02.038.

6. Dorjsembe B, Lee HJ, Kim M, Dulamjav B, Jigjid T, Nho CW. Achilleaasiatica extract and its active compounds induce cutaneous wound healing. J Ethnopharmacol. 2017; 206:306–314. doi: 10.1016/j.jep.2017.06.006.

7. Singh H, Ali SS, Khan NA, Mishra A, Mishra AK. Wound healing potential of Cleome viscosa Linn. Seeds extract and isolation of active constituent. S Afr J Bot. 2017; 112: 460–465. doi: 10.1016/j.sajb.2017.06.026.

8. Muthu C, Ayyanar M, Raja N, Ignacimuthu S. Medicinal plants used by traditional healers in Kancheepuram District of Tamil Nadu, India. J EthnobiolEthnomed. 2006; 2(1):43. doi: 10.1186/1746-4269-2-43.

9. Kirtikar KRBB, Basu BD. Indian medicinal plants. Indian Medicinal Plants. 2. Dehra Dun: K.A. Longman; 1935. pp. 1294–1295.

10. Jayasinghe ULB, Jayasooriya CP, Bandara BMR, Ekanayake SP, Merlini L, Assante G. Antimicrobial activity of some Sri Lankan Rubiaceae and Meliaceae. Fitoterapia. 2002; 73(5):424–427. doi: 10.1016/S0367-326X(02)00122-3

11. Kumaresan PT, Saravanan A. Anticonvulsant activity of Morinda tinctoria Roxb. Afr J Pharm Pharmacol. 2009;3(2):063–065. [Google Scholar]

12. Deepti K, Umadevi P, Vijayalakshmi G. Antimicrobial activity and phytochemical analysis of Morinda tinctoria Roxb leaf extracts. Asian Pac J Trop Biomed. 2012; 2(3):S1440–S1442. doi: 10.1016/S2221-1691(12)60433-X. [CrossRef] [Google Scholar]

13. Subramanian M, Balakrishnan S, Chinnaiyan SK, Sekar VK, Chandu AN. Hepatoprotective effect of leaves of Morinda tinctoria Roxb. againstparacetamol induced liver damage in rats. Drug Invent Today. 2013; 5(3):223–228. doi: 10.1016/j.dit.2013.06.008.

14. Sreena KP, Poongothai A, Soundariya SV, Srirekha G, Santhi R, Annapoorani S. Evaluation of in vitro free radical scavenging efficacy of different organic extracts of Morinda tinctoria leaves. Int J Pharm Pharm Sci. 2011; 3(Suppl 3):207–209.

15. Amerasan D, Murugan K, Panneerselvam C, Kanagaraju N, Kovendan K, Kumar PM. Bioefficacy of Morinda tinctoria and Pongamiaglabra plant extracts against the malaria vector Anopheles stephensi (Diptera: Culicidae) J EntomolAcarol Res. 2015;47(1):31–40. doi: 10.4081/jear.2015.1986.

16. Sivakumar T, Sivamaruthi BS, Priya KL, Kesika P, Chaiyasut C. Evaluation of bioactivities of Morinda tinctoria leaves extract for pharmacological applications. Evaluation. 2018; 11(2):100–105.

17. P. Thirupathy Kumaresan and A. Saravanan. Anticonvulsant activity of Morinda tinctoria-Roxb. African Jr of Pharmacy and Pharmacology. 2009; 3(2):063-065.

18. Subramaniyan Vijayakumar., Srinivasan Prabhu, and Sellen Chandrasekhar. Anti-Inflammatory activity of Morinda tinctoria on Carrageenan induced Rat Paw Edema. Int J Med Biosci. 2012; 1(4): 55 – 60.

19. Anand M, Muralidharan P. Antioxidant activity of Methanolic fruit extract of Morinda tinctoriaRoxb in Cerebral Ischemia Induced by Bilateral Common Carotid artery occlusion in rats. American Journal of Pharmtech Research 2014; 4(2):1-14.

20. Isha I. Sait, Jyoti Harindran, A. Abdul Vahab, Jeena J. L, Nasli S. Jolly John. Potential hepatoprotective effect and antioxidant role of methanol extract of Morinda tinctoria in carbon tetrachloride induced hepatotoxicity in albino rats. Int J Pharm. 2014; 4(1): 363-368.

21. Kanchanapoom T. Iridoid and phenolic glycosides from Morinda coreia. Phytochemistry. 2001; 59(5); 551-556. doi: 10.1016/s0031-9422(01)00426-5

22. Menone ML, Pesce SF, Diaz MP, Moreno VJ, Wunderlin DA. Endosulfan induces oxidative stress and changes on detoxification enzymes in the aquatic macrophyte Myriophyllumquitense. Phytochemistry. 2008; 69:1150-1157.

23. Ghasanfari G, Minaie B, Yasa N, Leilu AN, Azadeh M. Biochemical and histopathological evidences for beneficial effects of SaturejaKhuzestanicaJamzad essential oil on the mouse model of inflammatory bowel diseases. Toxicol Mech. 2006; 16:365-372

24. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of aging. Nature. 2000; 408:239-247.

25. Hartwell JL. Plants used against cancer. Quarterman: Lawrence, MA; 1982.

26. Mariappan V, Shanthi G, Saridha D. Pharmacognostical Studies on Morinda tinctoria. Roxb. Int J Pharm Pharm Sci. 2012; 4(2): 636-638.

27. Atish K Sahoo, Nisha Narayanan, N Satheesh Kumar, S Rajan, and Pulok K Mukherjee. Phytochemical and therapeutic potentials of Morinda tinctoriaRoxb. (Indian mulberry). Oriental Pharmacy and Experimental Medicine. 2009; 9(2): 101-105.

28. Ruch RJ, Cheng SJ, Klaunig JE. Prevention of Cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinigenesis. 1989; 10: 1003-8.

29. Prieto P, Pineda M, Aguilar M. Spectrophotometric quantitation of antioxidant capacity through the formation of a Phoshomolybdenum complex: Specific application to the determination of vita min E. Analytical Biochemistry. 1999; 269:337–341.

30. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and Cytotoxicity assays. Journal of Immunological Methods. 1983; 65: 55-63.

31. Monks, A., Scudiero, D., Skehan, P., Shoemaker, R., Paull, K., Vistica, D., Hose, C., Langley, J., Cronise, P., Vaigro-Wolff, A., Gray-Goodrich, M., Campbell, H., Mayo, J., Boyd, 1991. Feasibility of high flux anticancer drug screen using a diverse panel of cultured human tumour cell lines. Journal of the National Cancer Institute. 83; 757-766.

32. Pooja A.S Bawa and Farhath Khanum. Anti-inflammatory activity of Rhodiolarosea- a second generation adaptogen. Phytother. Res. 2009; 23: 1099-1102.

33. Laouer H, Meriem, EK, Parado S, Baldovini N. An antibacterial and antifungal phenylpropanoid from C. monataum. Phytother Res. 2009; 23: 1726-30.

Received on 15.12.2023 Modified on 08.02.2024

Res. J. Pharmacognosy and Phytochem. 2024; 16(2):83-88.

DOI: 10.52711/0975-4385.2024.00016

Treatment	Dose (mg/kg)	Paw volume (ml) as measured by mercury displacement at 6 hour	Percentage inhibition of paw edema
Group I Normal saline	10ml/kg orally	5.85±0.96	-
Group II Standard	100mg/kg I.P. Diclofenac sodium	1.70±0.42	71.00 % *a
Group III Treatment Control	100mg/kg EAEMC	2.23±0.58	69.00 % *a
Group IV Treatment Control	100mg/kg EEMC	1.86±0.52	70.00 % *a
Group V Treatment Control	100mg/kg AEMC	1.78±0.56	62.00 % *a