INTRODUCTION:

Diabetes is a serious and widespread metabolic disease. Generally, it is characterized by a decrease in the insulin level or increase in the insulin resistance. Persistent high glucose levels may lead to damage to various organs, particularly, eyes, cardiovascular system, nervous system and kidneys. Numerous drugs are available in the market for stabilizing the blood sugar levels. These drugs besides having potent actions, have several side effects as well^1,2. Natural products can be a good alternative to modern day medicine having an advantage of being safe and economical^3-7.

Pyrexia or fever is the increase in the body temperature above the normal ranges. Fever may be a sign of an underlying infection or some other disease. Apart from eliminating the possible cause of fever it is also necessary to bring the temperature of the body to normal so as to improve the patient’s condition.

Conventional antipyretic therapies are notorious for causing GIT and renal problems. Plant based drugs have fewer side effects than the currently available NSAIDS in the market and may also serve to be an economical alternative in developing countries^8,9.

Breynia disticha (also known as snowbush) belongs to the family Phyllanthaceae. It exists in the form of shrub or small trees. The plant is generally used for ornamental purposes due to its colorful leaves and the ability spread rapidly and cover the walls. Traditionally, the pant has been utilized for its pain relieving ability. In addition, it might be assumed that the plant may possess additional medicinal activities due to presence of a variety of secondary metabolites^10,11. Previously, the plant has been studied for enzyme inhibition, anticancer, bronchorelaxant and anti-inflammatory activities using in-vitro assays and in-vivo mice models^12,13. Vernonia elaeagnifolia belongs to family Asteraceae. It is considered as a creeper due to its ability to curl upwards and attach to solid surfaces. Due to this reason the plant is often grown in houses for screening purposes. Traditionally, the plant is known for its leech repellant activity¹⁴. The plant has been studied for antiulcer, antihyperlipidemic, cytotoxic and anti-inflammatory activities^15-17.

Surprisingly, both plants are relatively less studied particularly in terms of their chemical composition. Most bioactivities are preliminary and recent. This makes these plants an interesting subject for phytochemists and ethnopharmacologists. The current works brings a deeper insight into the medicinal properties of the plants with special reference to lower mammals (rabbits) as compared to the previous studies conducted on rodents (mice). The concept of polyherbal formulations has also been implemented highlighting the compatibility of the extracts together with the synergistic effects. Herein, we report the irritant, hypoglycemic and antipyretic activity of the plant extracts on Oryctolagus coniculus. The results suggest the plants to be of significant medicinal value.

MATERIAL AND METHODS:

Plant material:

The aerial parts of Breynia disticha and Vernonia elaeagnifolia were collected from Arain plant nursery, Lahore during the spring season and were authenticated by Dr. Muhammad Ajaib, GC University Lahore, Pakistan as B. disticha and V. elaeagnifolia. The plant samples were deposited at Herbarium of GC University.The voucher number for B. distichaand V. elaeagnifolia were 2282 and 2283 respectively.

Extraction:

The fresh plant material was washed carefully under tap water to remove contamination. It was then dried under shade at room temperature for three weeks and carefully ground into coarse powder while ensuring low temperature during entire process. The plant material was extracted by maceration using methanol (1000 ml twice for 500gm of powder). The extract was filtered through muslin cloth and concentrated using rotary evaporator (Heidolph 4000) at 35ºC and finally dried in desiccator under vacuum. The extraction of B.disticha and V.elaeagnifolia yielded 48.3g and 56.1g of crude extract respectively.

Chemicals:

Methanol, acetone and aspirinwere purchased from Sigma Aldrich, USA. Glibenclamide was purchased from Tocris Bioscience, UK. All other chemicals were of analytical grade.

Experimental Animals:

Adult healthy male rabbits (O. coniculus) weighing 1.8-2.0Kg were obtained from a local market in Lahore. The animals were allowed to acclimatize for two weeks during which they were kept at room temperature and provided controlled light and dark cycles each of 12 hours. The animals were fed freely with a mixture of green fodder, banana, cauliflower and cabbage and tap water ad libitum. The study protocol was approved by the ethical committee (AREC016-13) of the Faculty of Pharmacy, The University of Lahore.

Irritant activity:

Sample solutions (w/v) were prepared by dissolving 50mg of plant extract in 50ml of acetone. Acetone served as control. The rabbits were distributed randomly into 8 groups (n=6). Group I, II, III and IV received 20µl, 40µl, 80µl and 120µl of BME while Group V, VI, VII and VIII received 20µl, 40µl, 80µl and 120µl of VME respectively. Hairs from the inner surface of rabbit’s ear were removed with the help of razor. One ear served as control while second as experimental. 20, 40,80, and 120μl solution of each plant extract was applied to experimental ear of each respective group while a same volume of neat solvent was applied to the corresponding control ear. After 30minutes the ears were observed for redness. The rabbits were kept under observation for 24 hours and periodically checked with the intervals of 30 minutes for any irritant effect. Irritancies on rabbit ears were recorded according to the scale of Hacker^18,19.

Antipyretic activity:

Antipyretic activity was studied using boiled milk induced pyrexia model in experimental rabbits²⁰. The extracts at a concentration of 200mg/kg were administered to the rabbits orally. The synergistic effect of the two extracts (BME and VME as 100+100mg combined) was also studied. Group I (standard or positive control) received aspirin (10mg/kg) whereas group II received vehicle only and served as negative control. Group III and IV received 200mg/kg of BME and VME respectively whereas group V was administered with a 200mg/kg combined extract. Each group comprised of 6 adult healthy rabbits of either sex with weight ranging from 1-1.5kg.

Hypoglycemic activity:

Hypoglycemic activity was determined according to a previously published method with some minor modifications^21,22. The rabbits were randomly divided into 5 groups (n=6). Group I served as positive control (standard) group and received 5mg/kg glibenclamide whereas group II (negative control) received the vehicle (distilled water) only. Group III and IV received 500mg/kg BME and VME respectively whereas group V received a mixture of both (250mg BME+250mg VME). Blood sugar level was determined at the fasting stage and at 2, 4, 6 and 8hour intervals after the administration of the samples.

Statistical analysis:

The data was expressed as mean ± standard deviation (SD) and analyzed using analysis of variance (ANOVA). A probability value of p<0.05 was considered as statistically significant.

RESULTS AND DISCUSSION:

The crude extracts of BME and VME at the concentration of 20μg/20μl, 40μg/40μl, 80μg/80μl and 120μg/120μl were applied on the shaved inner surface of rabbit’s ear and observed for the period of 24 hours. No signs of redness or edema were observed at any of the concentrations used for both plants (table 2). The results show that both BME and VME have no irritant effects at low and high doses.

The Hacker's recommended scale (table 1) offers a practical way to record and rate the degree of irritation caused by various compounds on skin surfaces¹⁸. Plant extracts exhibiting irritant effect on cutaneous surfaces might not prove to be feasible for topical applications due to irritation and discomfort. In this context, having no irritant effect is thus beneficial providing an option for an additional route of administration.

The results of antipyretic activity are mentioned in table 3. The rectal temperature in case of control group (solvent only) remained almost steady and no significant decrease was noted. The standard group (aspirin) showed significant results at all three time intervals with a maximum percentage reduction of 93.7% (P<0.001). The results of the polyherbal mixture resembled closely to that of aspirin which exerted pronounced antipyretic effect. The results were found to be significant at each time interval with maximum reduction of 93.1% (P<0.001). BME and VME groups also produced significant results with 84% (P<0.001) and 75.1% (P<0.01) reduction at the end of the study. The results show both plants and their mixture to be of high antipyretic activity. It is noteworthy that the pyrexia was induced using boiled milk. Nevertheless, many antipyretic studies show the use of yeast for the induction of pyrexia^23-25. Milk-induced pyrexia may offer a more stable pyretic model due to the presence of multiple microbial strains,which,in contrast to a single dominant yeast species, might prolong the immune response and ensure delayed amelioration of pyrexia²⁶.

Table 1: Irritant scale grading

Reaction Grades	Explanation
-	No reaction
±	Doubtful reaction, diffused inflammation with no clear visible symptom
+	Slight reddening of the main vessels without reddening of the area in between
++	Marked reddening of the main vessels with reddening of the area in between
+++	Intense reddening of the entire ear often accompanied with macroscopic visible hyperplasia
++++	Visible exudative lesion with marked epidermal damage

Table 2: Irritant activity of plant extracts

Group No.	Extract.	Concentration	Response time (hours)
Group No.	Extract.	Concentration	1	2	3	4	5	6	7	8	9	10	12	24
1.	BME	20µl	-	-	-	-	-	-	-	-	-	-	-	-
2.		40µl	-	-	-	-	-	-	-	-	-	-	-	-
3.		80µl	-	-	-	-	-	-	±	±	-	-	-	-
4.		120µl	-	-	-	-	-	±	±	±	±	-	-	-
5.	VME	20µl	-	-	-	-	-	-	-	-	-	-	-	-
6.		40µl	-	-	-	-	-	-	-	-	-	-	-	-
7.		80µl	-	-	-	-	-	-	-	-	-	-	-	-
8.		120µl	-	±	±	±	±	-	-	-	-	-	-	-

BME Breynia distichia methanolic extract; VME Vernonia elaeagnefolia methanolic extract.

Table 3: Antipyretic activity of crude plant extracts

Sr. No	Groups	Dose	Rectal temperature before treatment “⁰C”		Rectal temperature after treatment “⁰C”
Sr. No	Groups	Dose	Normal (A)	3 hrs after injecting boiled milk (B)	After 1 hr (C-1)	After 2 hr (C-2)	After 3 hr (C-3)
1.	Control	2ml/kg	38.1±0.1	39.9±0.09	39.8±0.06 (7.1±1.22)	39.7±0.03 (10.8±2.66)	39.7±0.07 (14.7±2.22)
2.	Standard	10mg/kg	38±0.12	39.6±0.27	39.1±0.21 (31.3±2.02)*	38.6±0.3 (66.9±1.33)**	38.1±0.12 (93.7±0.73)***
3.	BME	200mg/kg	38.1±0.1	39.6±0.25	39.5±0.22 (8.8±0.55)	39±0.32 (44.7±1.46)*	38.3±0.09 (84.0±1.16)***
4.	VME	200mg/kg	38.3±0.38	39.8±0.2	39.5±0.15 (16.5±1.42)	39.3±0.17 (33.7±2.13)*	38.6±0.34 (75.1±1.56)**
5.	BME+VME mixture	100+100mg/kg	38.1±0.06	40.0±0.2	39.5±0.24 (30.8±1.63)*	38.8±0.06 (63.1±1.35)**	38.2±0.03 (93.1±1.61)***

BME Breynia distichia methanolic extract; VME Vernonia elaeagnefolia methanolic extract; All values are expressed in mean ± SEM (n=6); Percentage reduction in rectal temperature is given within parentheses; * significant (p<0.05); ** moderately significant (p<0.01); *** highly significant (p<0.001).

Table 4: Antidiabetic activity of crude plant extracts

Sr. No	Groups	Dose	Fasting blood glucose level (mg/dl) at different time intervals (hours)
Sr. No	Groups	Dose	0	2	4	6	8
1.	Control	2ml/kg	90.2±1.4	88.4±1.3 (2.0±0.2)	86.4±1.6 (4.2±0.3)	86.2±1.4 (4.4±0.1)	84.2±1.4 (6.7±0.1)
2.	Standard	5mg/kg	98.8±2.2	89.2±2.1 (9.7±0.2)	77.4 ±2.6 (21.7±1.0)**	59.0±1.6 (40.3±0.6)***	60.8±2 .0 (38.5±0.9)***
3.	BME	500mg/kg	101.0±2.4	93.8±1.9 (7.1±0.4)	83.8±1.8 (17.0±0.3)*	63.8±1.3 (36.8±0.6)**	62.8±1.2 (37.8±0.4)***
4.	VME	500mg/kg	94.0±2.0	91.0±2.0 (3.2±0.1)	80.0±1.9 (14.9±0.4)*	62.8±1.6 (33.2±0.3)**	61.0±1.2 (35.1±0.6)**
5.	BME+VME mixture	250+250 mg/kg	102.8±1.9	93.0±1.8 (9.5±0.2)	79.8 + 1.5 (22.4±0.5)**	61.8 + 1.3 (39.9±0.6)***	59.8+ 1.2 (41.8±0.9)***

BME Breynia distichia methanolic extract; VME Vernonia elaeagnefolia methanolic extract; Values are shown as mean fasting blood glucose level mg/dl ± S.E.M (n=6); Percentage reduction in blood glucose level is given within parentheses; * significant (p<0.05); ** moderately significant (p<0.01); *** highly significant (p<0.001).

The results of antidiabetic activity are given in table 4. The control group administered with the vehicle only, showed minor variation in the blood glucose level during the study period (maximum percentage reduction recorded to be 6.7%). All other groups started showing significant results from 4-hour interval. The maximum reduction was noted in the standard and the polyherbal group (P<0.01). Highly significant results (P<0.001) were recorded in the polyherbal, standard and BME groups at the end of the study (8^th hour). The highest reduction was noted in polyherbal group (41.8%). The results showcase the high antidiabetic activity of both plants as well as their mixture which is comparable to that of standard glibenclamide.

Based on the profound effect on the glucose profile of the test animals it can be safely assumed that the original active secondary metabolites responsible for the activity must have superior action when in isolated pure form as compared to the commercially marketed drugs. These natural products can provide a safe and cost effective alternative to the conventional antidiabetic therapies.

CONCLUSION:

The studies conclude that both plants and their mixture possess significant biological activities. Isolation of the compounds responsible for these activities is needed. Moreover, there is a need for an extensive study on the phytoconstituents from both plants as this can lead to promising lead compounds for future drug development.

ACKNOWLEDGEMENT:

We would like to thank Arain plant nursery for providing us the plant material.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCE:

1. Banday MZ, Sameer AS, Nissar S. Pathophysiology of diabetes: An overview. Avicenna Journal of Medicine. 2020; 10: 174-88. doi: 10.4103/ajm.ajm_53_20

2. Misra A, Gopalan H, Jayawardena R, Hills AP, Soares M, Reza‐Albarran AA, Ramaiya KL. Diabetes in developing countries. Journal of diabetes. 2019; 11(7): 522-539. doi:10.1111/1753-0407.12913

3. Panchal I, Panigrahi B, Patel CN. Recent Advancement towards Treatment of Diabetes. Research Journal Pharmacology and Pharmacodynamics. 2010; 2 (1): 12-22.

4. Preethi PJ. Herbal Medicine for Diabetes Mellitus: A Review. Asian Journal of Pharmaceutical Research. 2013; 3 (2): 57-70.

5. Jha AK, Verma P, Prasad M, Sachan NK, Gautam K. Alternative Medicine: An Introduction and Market Potential. Research Journal Pharmacognosy and Phytochemistry. 2009; 1(3): 146-152.

6. Sharma S, Sharma R, Singh H, Nelkumar B. History of Medicine – Herbal Medicine Scenario: A Review. Research Journal Pharmacology and Pharmacodynamics. 2011; 3 (2): 45-47.

7. Jadhav CA, Vikhe DN, Jadhav RS. Global and Domestic Market of Herbal Medicines: A Review. Research Journal of Science and Technology. 2020; 12(4): 327-330. doi: 10.5958/2349-2988.2020.00049.2

8. Cock IE, Luwaca N, Van Vuuren SF. The traditional use of Southern African medicinal plants to alleviate fever and their antipyretic activities. Journal of Ethnopharmacology. 2023; 303: 115850-61. doi: 10.1016/j.jep.2022.115850

9. Nilima T, Pranali S, Madhura T. Medicinal plant as a source of Antipyretic drug: A Review. Asian Journal of Pharmacy and Technology. 2021; 11(1): 84-87. doi: 10.5958/2231-5713.2021.00014.3

10. El-Banna H, Haroun SA, Albishi TS, Rashed AA, Albadrani M, Abdelaal K, Abdou AH. The Natural Alternatives: The Impact of Plant Extracts on Snowbush (Breynia disticha Forst.) Cuttings’ Morpho-Physiological and Biochemical Characteristics. Horticulturae. 2023; 9(10): 1122-1135. doi:10.3390/horticulturae9101122

11. Saadullah M, Asif M, Arif S, Kanwal B. A Comprehensive Review on Traditional Uses, Chemical Constituents, and Diverse Pharmacological Importance of the Genus Breynia. Records of Natural Products. 2022; 6: 538-549. doi: 10.25135/rnp.314.2112.2280

12. Nakashima KI, Abe N, Oyama M, Murata H, Inoue M. Sulfur-containing spiroketals from Breynia disticha and evaluations of their anti-inflammatory effect. Beilstein Journal of Organic Chemistry. 2023; 19(1): 1604-1614. doi: 10.3762/bjoc.19.117

13. Saadullah M, Asif M, Uzair M, Afzal S, Rashid SA, Rashad M, Mahmood S. Pharmacological evaluation of the hypoglycemic and anti-Alzheimer’s activities of aerial parts of Breynia distachia (Phyllanthaceae). Tropical Journal of Pharmaceutical Research. 2022; 21(3): 579-587. doi: 10.4314/tjpr.v21i3.18

14. Vongsombath C, De Boer HJ, Pålsson K. Keeping leeches at bay: Field evaluation of plant-derived extracts against terrestrial blood-sucking leeches (Haemadipsidae) in Lao PDR. Acta tropica. 2011; 119(2): 178-182. doi: 10.1016/j.actatropica.2011.05.014

15. NawaleS, Priyanka N, Das S, Raju MG. Data of in vivo screening of antiulcer activity for methanolic extract of Vernonia elaeagnifolia DC. Data in brief. 2019; 23: 103753. doi: 10.1016/j.dib.2019.103753

16. Acharzo AK, Rahman S, Anisuzzman M, Islam MA, Kundu P, Bokshi B, Billah M. Antihyperglycemic, Neuropharmacological, Cytotoxic, Anticoagulant, and Anti-inflammatory Pharmacological Evaluations of Vernonia elaeagnifolia DC Leaves Secondary Bioactive Metabolites. European Journal of Pharmaceutical Research. 2023; 3(4): 1-9. doi: 10.24018/ejpharma.2023.3.4.70

17. Sultana A, Khaliq T, Rehman AU, Majeed W, Faisal MN, Aslam B, Anwar H. Pharmacological evaluation of Vernonia elaeagnifolia (Asteraceae) leaves in hyperlipidemic albino rabbits. Tropical Journal of Pharmaceutical Research. 2017; 16(5): 1077-1083. doi; 10.4314/tjpr.v16i5.15

18. Hacker E. Methods of Cancer Research. Academic Press, London.1971.

19. Saeed-ul-Hassan S, Khalil-ur-Rehman M, Niaz U, Saeed MA., Hussain K, Rao SA, Ahmed I. Isolation and characterization of irritant components of Euphorbia pilulifera L. Pakistan Journal of Pharmaceutical Sciences. 2013; 26(1): 31-37.

20. Ahmed S, MaalikS, Zehra T, Baig SG, Zehra S. Preclinical assessment of analgesic, anti-inflammatory and antipyretic potential of Fragaria ananassa and Actinidia deliciosa fruit extract. Pakistan Journal of Pharmaceutical Sciences. 2021; 34(2): 521-527.

21. Sudasinghe HP, Peiris DC. Hypoglycemic and hypolipidemic activity of aqueous leaf extract of Passiflora suberosa L. PeerJ. 2018; 6: 4389. doi: 10.7717/peerj.4389

22. Kumbhare M, Sivakumar T, Surana A. Evaluation of Hypoglycemic potential of Moringa oleifera bark extracts on normal and Alloxanized diabetic rats. Research Journal of Pharmaceutical Dosage Forms and Technology. 2021; 13 (2): 95-9. doi: 10.52711/0975-4377.2021.00017

23. Shrivastava A, Parveen N, Dewangan G, Vinay VS, Bhattacharya A, Sharma M. Evaluation of Anti-Pyretic Potential of Cassia alata Leaves. Asian Journal of Pharmaceutical Research. 2014; 4 (4): 195-197.

24. Elumalai A, Eswaraiah MC, Lahari KM, Shaik HA. In-vivo screening of Bougainvillea glabra leaves for its Analgesic, Anti-pyretic and Anti-inflammatory activities. Asian Journal of Research in Pharmaceutical Science. 2012; 2 (3): 85-87.

25. Ghiwarem NB, Nesari TM. Antipyretic Activity of Piper nigrum and Nyctanthes arbor-tristis in Different Dosage Forms. Research Journal of Pharmacy and Technology. 2010; 3 (1): 157-160.

26. Quigley L, McCarthy R, O'Sullivan O, Beresford TP, Fitzgerald GF, Ross RP, Cotter PD. The microbial content of raw and pasteurized cow milk as determined by molecular approaches. Journal of Dairy Science. 2013; 96 (8): 4928-4937.

Received on 05.01.2024 Modified on 27.03.2024

Research J. Pharm. and Tech 2024; 17(8):3658-3662.

DOI: 10.52711/0974-360X.2024.00570