Phytochemical, In vitro Anti-inflammatory and Antimicrobial Potential of Hugonia mystax L.

 

K.P. Jaiganesh1*, T.J. Jasna2, A.C. Tangavelou3

1Division of Pharmacognosy and Phytochemistry Research Laboratory, Dhanalakshmi Srinivasan College of Pharmacy, Perambalur - 621 212, Tamil Nadu, India.

2Department of Pharmacognosy and Phytochemistry, Nehru College of Pharmacy, Pampady, Thiruvilwamala, Thrissur - 680 588, Kerala, India.

3Department of Plant Biology and Plant Biotechnology, Guru Nanak College (Autonomous), Velachery, Chennai 600042, Tamil Nadu, India.

*Corresponding Author E-mail: kpjaiyaganesh@gmail.com

 

ABSTRACT:

Hugonia mystax L., (Linaceae), is commonly distributed in the thorny scrubs and tropical dry evergreen forests of Tamil Nadu, which has been valued for centuries in traditional system of medicine for the treatment of various ailments. In the present study was an attempt to investigate the phytochemical nature and anti-inflammatory, antimicrobial potential by adopting suitable methods. Phytochemical analysis of Hugonia mystax L., plant extracts revealed the presence of various biochemical compounds such as alkaloids, flavonoids, glycosides, triterpenoids and saponins etc. Since triterpenoids and flavonoids have remarkable anti-inflammatory activity, so our present work aims at evaluating in vitro anti inflammatory activity of Hugonia mystax L., by HRBC membrane stabilization method. The inhibition of hypotonicity induced HRBC membrane lysis was taken as a measure of the anti-inflammatory activity. The percentage of membrane stabilization for ethanolic extracts and Diclofenac sodium were done at different concentrations. The maximum membrane stabilization of Hugonia mystax L., extracts was found to be 94.97 % at a dose of 2000 μg/ml. Therefore, our studies support the isolation and the use of active constituents from Hugonia mystax L., in treating inflammations.

 

KEYWORDS: Anti-inflammatory, Diclofenac sodium, Hugonia mystax L., Human Red Blood Cell (HRBC), Membrane stabilization.

 

 


INTRODUCTION:

The nature has provided abundant plant wealth for all the living creatures, which possess medicinal virtues. Therefore, there is a necessity to explore their uses and to ascertain their therapeutic properties. Inflammation is a complex reaction to injurious agents, such as microbes and damaged usually necrotic cells that consist of vascular responses, migration and activation of leucocytes, and systemic reaction. Inflammatory reaction may cause hypersensitivity reaction to insect bites, drugs and toxin. Inflammatory reaction underlies some common chronic diseases such as rheumatoid arthritis, atherosclerosis and lung fibrosis1. Inflammation is classified as Acute and Chronic. Acute inflammation is an initial response of the body to harmful stimuli, achieved by the increased movement of plasma and leukocytes from the blood into injured tissues. A cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, immune system, and various cells within injured tissue. Chronic inflammation is a prolonged inflammation, leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of tissue from the inflammatory process 2.

 

The genus Hugonia L. of family Linaceae comprise about 40 species in the world; of which Hugonia mystax L. was reported from India3,4. This plant Hugonia mystax L. is locally known as “Modirakanni”. Ethnobotanically, the fruits are used by the tribals of Kalakad Mundanthurai for the treatment of Rheumatism5. Roots were used as anthelmintic, astringent and also used for dysentery, snake bite, fever, inflammation and rheumatism conditions. Biological activities such as analgesic, anti-inflammatory and ulcerogenic were also reported6-9. Roots of Hugonia mystax L. were evaluated for preliminary phytochemical screening and antimicrobial activity. Preliminary phytochemical screening showed the presence of various classes of secondary metabolites such as flavonoids, phenols, saponins, steroids, tannins and terpenoids. Antimicrobial activity of petroleum ether, chloroform, ethanol and aqueous root extracts showed significant activity against various human pathogens 10.

 

MATERIALS AND METHODS:

The plant materials (leaves, stem, root and fruits) of Hugonia mystax L. were collected from the Marakanam forest vicinity, of Villupuram district, Tamil Nadu. The collected plant materials was botanically identified and confirmed by using local flora such as Flora of Tamil Nadu Vols. 1-3 Hugonia mystax L.11 and An Excursion Flora of Central Tamil Nadu, India12. The fresh plant materials were collected and then morphological features of the specimen were studied directly in the field and were photographed. Fresh parts of plant materials, such as leaves and stems are collected and kept separately in polythene bags. These materials are dried under shade separately in the laboratory for 3 to 4 days and the dried parts are stored in dry polythene bags for carrying out phytochemical and biological investigations.

 

Preparation of extracts:

The collected leaf material were cut into small pieces, shade dried and coarsely powdered by using a pulverizor. Then, the leaf powder was subjected to successive solvent extraction with organic solvents of increasing polarity such as petroleum ether, chloroform and ethanol by continuous hot percolation method using soxhlet apparatus13 and aqueous (0.25% v/v of CHCl3 in water) extract was also prepared by cold maceration method. Then, the extracts were collected and distilled off on a water bath at atmospheric pressure and the last trace of the solvent was removed in vaccuo. The resulted extracts were used for preliminary phytochemical screening and antimicrobial activities.

 

Preliminary phytochemical screening:

All the extracts were subjected to preliminary phytochemical screening followed by the standard methods 13, 14.

 

Antimicrobial Activity15

Ethanolic extract of the selected plant was used to prepare various concentrations such as 75, 50, 25mg/ml and ethanolic leaf extract. These were used for the screening of antimicrobial potential compared with respective standard antibiotic (10µg/ml).

 

Test Microorganisms:

The following bacterial and fungal strains were used for the screening of antimicrobial activity. All the microbial strains of human pathogens used were procured from IMTECH, Chandigarh and the procured microbes are the Gram-negative bacteria, such as Escherichia coli (MTCC 724), Staphylococcus aureus (MTCC 96), and fungi such as Candida albicans (MTCC 227) respectively.

 

Determination of antimicrobial activity

Agar well‐diffusion method16 was followed to determine the antimicrobial activity. Nutrient agar (NA) and Potato Dextrose Agar (PDA) plates were swabbed (sterile cotton swabs) with 8 hours old‐broth culture of respective bacteria and fungi. Two wells (10mm diameter) were made in each of these plates using sterile cork borer. About 0.3 ml of different concentrations (75, 50, 25 mg/ml) of plant extracts were added using sterilized dropping pipettes into the wells and allowed to diffuse at room temperature for 2 hours. The plates were incubated at 37°C for 18‐24 hours for bacterial pathogens and 28°C for fungal pathogens. Respective solvent control for leaf extracts was also maintained and the diameter of zone of inhibition was recorded in mm and compared with standard values. Triplicates were maintained and the experiment was repeated thrice and the average values were recorded for antimicrobial activity.

 

In vitro anti-inflammatory activity17

Preparation of Human Red Blood Cells (HRBC) Suspension

Fresh whole human blood was collected and mixed with equal volume of sterilized Alsever solution (2 % dextrose, 0.8 % sodium citrate, 0.05% citric acid and 0.42 % sodium chloride in water). Sodium oxalate was used to prevent clotting. All the blood samples were stored at 4C for 24 hours before use. The blood was centrifuged at 3000 rpm for 10 min and packed cells were washed three times with isosaline (0.85%, pH 7.2). The volume of the blood was measured and reconstituted as 10% v/v suspension with isosaline.

 

Heat Induced Hemolysis:

The principle involved here is stabilization of human red blood cell membrane by hypo tonicity induced membrane lysis. The assay mixture contains 1ml phosphate buffer [pH 7.4, 0.15 M], 2 ml Hyposaline [0.36 %], 0.5 ml HRBC suspension [10 % v/v] with 0.5 ml of plant extracts and standard drug Diclofenac Sodium of various concentrations (100, 250, 500, 1000 μg/ml) and control (distilled water instead of hypo saline to produce 100 % hemolysis) were incubated at 37oC for 30 min and centrifuged respectively. The hemoglobin content in the suspension was estimated using spectrophotometer at 560 nm. The percentage of hemolysis of HRBC membrane can be calculated as follows:

                                Optical density of Test sample

% Hemolysis =––––––––––––––––––––––––––––X 100

   Optical density of Control)

 

The percentage of HRBC membrane stabilisation can be calculated as follows:

 

                          100 –Optical density of Test sample

% Protection = ––––––––––––––––––––––––––––X 100

    Optical density of Control

 

RESULTS:

Preliminary phytochemical screening

The preliminary phytochemical screening (Table 1) showed the presence of phenolic compounds, steroids, saponins, tannins, flavonoids and alkaloids in ethanol extract. Amino acids, anthraquinones, coumarins and lipids were completely absent.

 

Table 1 Preliminary phytochemical screening of Hugonia mystax

Phytoconstituents

Ethanol extract

Powder

Alkaloids

-

-

Amino acids and Proteins

-

-

Anthraquinones

-

-

Carbohydrates

-

-

Catechins

-

-

Flavonoids

+

+

Glycosides

-

-

Gums and Mucilages

+

-

Phenolic compounds

+

+

Saponins

+

+

Steroids

+

+

Tannins

+

+

Terpenoids

+

+

(+) = Presence of phytoconstituents

(-) = Absence of phytoconstituents

 

Antimicrobial activity

The antimicrobial activity of ethanol extract of Hugonia mystax L. of 25, 50, 75 mg/ml is effective against microorganisms (Table 2). In the present investigation, ethanol extract with Ampicillin and Penicillin (10µg/ml) were exhibited potent antimicrobial activity against various species (Fig. 1).

 

Table 2 Antimicrobial activity of Hugonia mystax L.

Microorganisms

Zone of inhibition in mm

25 mg/ml

50 mg/ml

75 mg/ml

Standard

(10 µg/ml)

Escherichia coli

18

24

30

35 (A)

Staphylococcus aureus

17

25

30

35 (A)

Candida albicans

17

24

31

32 (P)

A – Ampicillin; P – Penicillin

 

Fig. 1: Antimicrobial activity of Hugonia mystax L. extracts against pathogens

 

In vitro anti-inflammatory activity

The inhibition of hypotonicity induced HRBC membrane lysis i.e, stabilization of HRBC membrane was taken as a measure of the anti inflammatory activity. The percentage of membrane stabilization for ethanolic extracts and Diclofenac sodium were done at 100, 250, 500, 1000 μg/ml. Ethanol extracts of Hugonia mystax L. are effective in inhibiting the heat induced hemolysis of HRBC at different concentrations (100 - 1000μg/ml) as shown in (Table 3). It showed the maximum inhibition 85.67% at 1000μg/ml (Fig. 2-4).


 

Table 3. Effect of Hugonia mystax and Standard on HRBC membrane hemolysis and membrane stabilization

Conc.

(μg/ml)

Absorbance

(560 nm)

% Hemolysis of sample

% Stabilisation of sample

% Hemolysis of Diclofenac sodium

% Stabilisation of Diclofenac sodium

Control

0.49±0.02

-

-

-

-

100

0.37±0.001

32.25

67.74

47.18

52.81

250

0.32±0.001

20.77

79.22

23.47

76.54

500

0.27±0.003

18. 68

81.32

16.05

84.05

1000

0.23±0.001

14.34

85.67

12.43

87.56

 


Fig.4: Anti-inflammatory activity by HRBC method

 

DISCUSSION:

Medicinal herbs represent a rich source from which novel antibacterial and antifungal chemotherapeutic agents may be obtained18,19. In recent years, interest in higher plant extracts exhibiting antimicrobial activity has increased20-22 claim to develop a novel lead against multidrug resistance microorganisms23. In the present study, all the tested extracts showed concentration-dependent activity against the tested organisms due to the presence of various phytoconstituents. Secondary metabolites in plant products are responsible for several biological activities in man and animals24. The active components usually interfere with growth and metabolism of microorganisms in a negative manner. Antimicrobial properties of several plant extracts have been attributed due to the secondary metabolites. The reason for the difference sensitivity between the gram-positive and gram-negative bacteria could be ascribed to the morphological differences between these microorganisms, gram-negative pathogens having an outer phospholipidic membrane carrying the structural lipopolysaccharide components. This makes the cell wall impermeable to lipophilic solutes, while porins constitute a selective barrier to hydrophilic solutes with an exclusion limit of about 600 Da. The gram positive bacteria should be more susceptible having only an outer peptido glycone layer which is not an effective permeability barrier. Phenolic content of plant extracts possess antimicrobial activity and highly oxidized phenols are more inhibitory because of phenolic toxicity to microorganisms. In addition, leaf extracts also possess antimicrobial potential against all pathogens which may be due to the presence of steroids and alkaloids25,26.

 

Various flavonoids (i.e.) quercetin, apigenin, tea catechins have also been shown to have anti-inflammatory activity by inhibiting cycloxygenase-2 (COX-2) and inducible nitric oxide synthase, which is related to antioxidant activity. Flavonoids also inhibit cytosolic and tyrosine kinase and also inhibit neutrophil degranulation27.

 

One of the major and well documented causes of inflammation is denaturation of proteins. The investigation is done on the mechanism of anti-inflammatory activity. The further study was carried out on the ability of extract to inhibit the protein denaturation as the part of investigation. For denaturation of albumin protein it seemed very efficient. Extracts were effectively inhibiting the heat-induced hemolysis28. Hypotonic solution has hemolytic effect. Hemolysis occurs when there is accumulation of excessive fluid into the cells resulting in rupture of RBC membrane. When the red blood cell membrane gets injured, it will make the cell more susceptible to secondary damage. This damage is occurred by free radical-induced lipid peroxidation29. The leakage of serum protein and fluids into the tissue can be prevented by membrane stabilization. This process goes on by inflammatory intermediators where there is an increase in permeability of membrane30.

 

HRBC method was adopted for the investigation of anti-inflammatory activity because erythrocyte membrane is analogous lysosomal membrane and its stabilization implies that the extract may as well stabilize lysosomal membranes. Stabilsation of lysosomal membrane is important in limiting the inflammatory response of lysosomal constituents of activated neutrophil, such as bactericidal enzymes and proteases, which cause further tissue inflammation and damage upon extra cellular release31.

 

The hypotonic solution has hemolytic effect. Hemolysis occurs when there is accumulation of excessive fluid into the cells resulting in rupture of the RBC membrane. When the red cell membrane gets injured, it will make the cell more susceptible to secondary damage. This damage is occurred by free radical-induced lipid peroxidation. The leakage of serum protein and fluids into the tissue can be prevented by membrane stabilization. This process go on by inflammatory intermediators where there is an increase in permeability of membrane. Ethanolic extract of Hugonia mystax extract might be stabilize the membrane of RBC by precluding the discharge of lytic enzymes and other active inflammatory mediators32.

 

The present investigation is concluded that ethanolic extract of Hugonia mystax are capable of inhibiting inflammatory reactions as well as pain as compared with reference standard. The results provided experimental voidance for its traditional use in treating various diseases associated with inflammation and pain.

 

ACKNOWLEDGEMENT:

The authors are highly thankful to The Founder- Chairman, Shri. A. Srinivasan, Dhanalakshmi Srinivasan College of Pharmacy, Perambalur, Tamil Nadu, and The Chairman and Managing Trustee, Adv. P. Krishna Das, LLB, MBA, BEM, Dr. P. Krishna Kumar, CEO & Secretary, Nehru College of Pharmacy, Pampady, Thiruvilwamala, Thrissur, Kerala, for providing all the facilities to carry out this study.

 

REFERENCES:

1.        James T. Wu, Lily L. Wu. Chronic systemic inflammation leading eventually to myocardial infarction, stroke, COPD, renal failure and cancer is induced by multiple risk factors. Journal of Biomedical and Laboratory Sciences (2007 Nov); 19 (3): 1-6.

2.        James T. Wu, Lily L. Wu. Acute and chronic inflammation: effect of the risk factor (s) on the progression of the early inflammatory response to the oxidative and nitrosative stress. Journal of Biomedical and Laboratory Sciences (2007 Nov); 19 (3): 71-74.

3.        Santapau H, Henry A.N. A dictionary of flowering plants in India. 4th ed. New Delhi: Council of Scientific and Industrial Research; 1983.103.

4.        Pullaiah T, Chennaiah E. Flora of Andhra Pradesh. 1st ed. Jodhpur, India: Scientific Publishers; Vol. 1, 1997.

5.        Sutha S. Mohan V.R, Kumaresan S, Murugan C, Athiperumalsami T. Ethnomedicinal plants used by the tribals of Kalakad Mundanthurai Tiger Reserve (KMTR), Western Ghats, TamilNadu, for the treatment of rheumatism. Indian Journal of Traditional Knowledge (2010 July); 9 (3): 502-509.

6.        Balasubramaniam P, Rajasekaran A, Prasad N. Folk Medicine of the Irulas of Coimbatore Forests. Ancient Science of Life (1997 Jan); 16: 1-3.

7.        Guha Bakshi D.N, Sensarma P, Pal DC. Lexicon of Medicinal Plants in India. 2nd ed. Calcutta, India: Naya Prokash; Vol. 2, 2001. 369.

8.        Rastogi P, Mehrotra B.N, Sinha S, Srivastava M, Bhushan B. Compendium of Indian Medicinal Plants. 1st ed. Lucknow: Central Drug Research Institute, Publications and Information Directorate; 4, 2002. 386.

9.        Yoganarasimhan S.N, Medicinal Plants of India. 1st ed. Bangalore, India: Interline Publishing Pvt. Ltd.; Vol. 2, 2000. 275.

10.      Vimalavady A, Kadavul K, Tangavelou A.C. Phytochemical screening and antimicrobial activity on the fruits of Hugonia mystax L. (Linaceae). International Journal of Pharmaceutical Sciences and Research (2012 Apr); 3 (4): 1178-1183.

11.      Nair N.C, Henry A.N. Flora of Tamil Nadu, India. Series I: Analysis. 1st ed. Coimbatore: Botanical Survey of India; Vol. 1, 1983. 188.

12.      Mathew K.M. An Excursion Flora of Central Tamil Nadu, India. New Delhi, India: Oxford & I.B. Publishing House Pvt. Ltd., 1991.

13.      Harborne J.B. Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. 1st ed. London: Chapman and Hall Ltd., 1973. 49-188.

14.      Trease G.E, Evans W.C. Pharmacognosy: 14th ed., London: W. B. Sounders Co. Ltd., 1996. 543.

15.      Purushotham K.G, Arun P, Johnsy Jayarani J, Vasnthakumari R, Sankar L, Bijjam A.V.I, Prakash Reddy. Synergistic in vitro antibacterial activity of Tectona grandis leaves with tetracycline. International Journal of PharmTech Research (2010 Jan-Mar); 2 (1): 519-523.

16.      Perez C, Paul M, Bazerque P. Antibiotic assay by agar-well diffusion method. Acta Biologiae et Medicinae Experimentalis (1990); 15: 113-115.

17.      Gandhidasan R, Thamaraichelvan A, Babura, S. Anti-inflammatory action of Lannea coromandelica by HRBC membrane stabilization. Fitoterapia (1991 Sep); 62 (1): 81‑83.

18.      Rates S.M.K. Plants as source of drugs. Toxicon. (2001 May) 39 (5): 603-613.

19.      Kilani-Jaziri S, Bhouri W, Skandrani I, Limem I, Chekir-Ghedira L, Ghedira, K. Phytochemical, antimicrobial, antioxidant and antigenotoxic potentials of Cyperus rotundus extracts. South African Journal of Botany. (2011 Aug); 77 (3): 767-776.

20.      Farnsworth N.R. Ethnopharmacology and drug development, In: Prance, G.T. [Ed.] Ethnobotany and the search for new drugs. Ciba Foundation Symposium -185, Chichester, Wiley, 1994, 42-59.

21.      Benzi G, Ceci A. Herbal Medicines in European Regulation, Phamacology Research (1997 May); 35 (5): 355-362.

22.      Bagghi A.K. Alternative medicine-old wine in a new bottle, Indian Medical Association (2000); 98: 332 -333.

23.      Othman M., Loh H.S, Wiart C, Khoo T.J, Lim K.H, Ting K.N. Optimal methods for evaluating antimicrobial activities from plant extracts. Journal of Microbiological Methods (2011 Feb); 84(2): 161-166.

24.      Sofowora A. Medicinal Plant and Traditional Medicine in Africa, 2nd ed., New York: John Wiley & Sons; 1986. 567.

25.      Kumar B, Sandhar H.K, Prasher S, Tiwari P, Salhan M, Sharma P. A review of phytochemistry and pharmacology of flavonoids. Internationale Pharmaceutica Sciencia, (2011 Jan-Mar); 1(1): 25-41.

26.      Jaiganesh K.P, Arunachalam G. preliminary phytochemical screening and antimicrobial potential of Pterospermum canescens, Roxb. (Sterculiaceae). (2011 Mar); 3(3): 139-141.

27.      Jaiganesh K.P, Arunachalam G. Screening of antimicrobial potential of Pterospermum canescens, Roxb. (Sterculiaceae) leaf and stem. International Journal of Research in Pharmacology and Pharmacotherapeutics. (2012 Aug); 1(2): 159-164.

28.      Nagababu E, Lakshmaiah N. Inhibition of microsomal lipid peroxidation and monooxygenase activities by eugenol. Free Radical Research, Taylor & Francis, 1994; 20: 253-66.

29.      Bukhari IA, Alhumayyd MS, Mahesar AL, Gilani AH. The analgesic and anticonvulsant effects of piperine in mice, Journal of Physiology and Pharmacology, 2013; 64(6): 789-794.

30.      Chaitanya R, Sandhya S, David S, Vinod B, Vinod KR Murali S. HRBC membrane stabilizing property of root, stem and leaf of Glochidion velutinum, International Journal of Research in Pharmaceutical and Biomedical Sciences, 2011; 2: 256-9.

31.      Chou C.T. The anti-inflammatory effect of Tripterygium wilfordii Hook F. on rats and inflammatory mediators release. Phytotherapy Research (1997 Mar); 11(2): 152- 154.

32.      Yesmin S, Paul A, Naz T, Atiqur Rahman A.B.M, Akhter S.F, Wahed M.I.I, Emran T.B, Siddiqui S.A. Membrane stabilization as a mechanism of the anti-inflammatory activity of ethanolic root extract of Choi (Piper chaba). Clinical Phytoscience (2020 Aug); 6 (59): 1-33.

 

 

 

Received on 21.07.2021         Modified on 13.08.2021

Accepted on 29.08.2021       ©A&V Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 2021; 13(4):169-173.

DOI: 10.52711/0975-4385.2021.00028