INTRODUCTION:

Medicinal plants, either as an extract, pure compound or as a derivative, present unlimited chances for the discovery of novel drugs. Most of the natural products used in folk medicine have solid scientific proof with an observation of their biological activities. However, there is modest information or proof available concerning the probable toxicity that medicinal plants may cause to the consumers. In relative to drug discovery and development, there are diverse weights of concern of all applicable groups such as health authorities, pharmaceutical industry, and patients which require to be taken into consideration.¹

Therefore, it should be highlighted that the conventional use of any plant for medicinal reasons, by no means, promises the safety of such a plant. This elevates concern about the potentially toxic effects resulting from the short-term and long-term use of such medicinal plants. The information of the acute and subchronic toxicity studies on medicinal plants or preparations derived from them should be fined to enhance the assurance in their safety to humans, mainly for use in the development of pharmaceuticals. Therefore, estimating the toxicological effects of medicinal plant extract planned to be used in animals or humans is a critical part of its assessment for possible toxic effects.²

Senecio is the major genus in the family Asteraceae and contained over 1500 species widespread all above the world. Recent studies indicated that numerous species of Senecio exhibit antimicrobial, antifungal and cytotoxic activities. Senecio edgeworthii hook Syn. Sonaki of the family Asteraceae is an erect herbaceous, much-branched herb distributed in the Kas plateau of Maharashtra India.^3,4,5.

Hence, in the existing work, Acute and subacute oral toxicity estimation of flower Oil, alcohol and Chloroform extracts of Senecio edgeworthii hook plant. No clinical proof or studies for the oral acute and sub-acute oral toxicity investigation for this plant are available in the literature. Therefore, acute and sub-acute oral toxicity studies were carried out by the OECD guidelines to verify and set up the safety for its use in clinical practice. The purpose of this study was to estimate the safety of Senecio edgeworthii hook in animals’ models.

MATERIALS AND METHODS:

Plant material:

The plant Senecio edgeworthii hook was collected in November 2020 from the area of Kas pathar, Satara Maharashtra, India the specimen was authenticated by comparing it with the voucher specimen deposited in the department of botany by Dr. Deshpande. S.M. Y.C.Institute of science, Satara Maharashtra, India.

Preparation of extract:

The plant material was dried in shade and powdered in the grinder. About 500 g of powdered plant material was extracted successively in the soxhlet apparatus by using solvents in order of increasing polarity viz., Pet. Ether, Chloroform and ethanol. After each extraction solvent was recovered using a rotary vacuum evaporator and dried extracts were stored in vacuum desiccators. These extracts were used toxicity study.^6,7,8

Extraction of Oil from SE flower:

Freshly collected Flower of the plant Senecio edgeworthii hook was dried under room temperature for three weeks. The dried flower material was made into a coarse powder and a weighed quantity of the powder was extracted in a soxhlet extractor with to which we added n-hexane for obtaining the total oil. During the extraction, the extracted lipids required the addition of 75% sodium chloride solution. The solvent was removed on a rotary evaporator at 50°C. Total oil was collected in a flask and stored at 4°C for further analysis^9,10

Animals:

Adult healthy albino mice weighing 25–30g and Wistar strain of rat either sex weighing from 150-200gm were used for the experiment. The animals were kept in polyacrylic cages with a wire mesh top and soft bedding. They were kept under standard husbandry conditions of 12h reverse light cycle with food and water ad libitum, maintained at temperature 22±2^oC. Approval of protocol the experimental protocol was approved by the Institutional Animal Ethics Committee of Satara College of Pharmacy, Satara and was carried out according to the CPCSEA (Committee for Control and Supervision on Experiments on Animals) guidelines for laboratory animal facilities. (Annexure-II) Protocol approval number Protocol No.: SCOP/IAEC/102/2020 (CPCSEA registration number- 1314/PO/Re/S/2009/CPCSEA)

Acute toxicity studies:

The acute oral toxicity study of extracts of SE- Oil, SE-Alcohol and SE-Chloro extracts of Senecio edgeworthii hook (SE) was estimated according to the OECD guideline 423 on mice (20–30g), where the maximum test dose of 5000mg/kg was used. All the animals were set aside at overnight fasting before every experiment with free excess to water. The animals were separated into four groups, each comprising 3 animals. The 1^st group, 2^nd, 3^rd and 4^th was considered as tested groups administrated orally extracts (dissolved in normal saline) extract at a dose of 300mg/kg, 1000mg/kg, 2000mg/kg and 5000mg/kg. Before the dose was received, the bodyweight of each animal was estimated and the dose was measured according to the body weight. The animals were observed for any toxic signs and symptoms for the first 4 h after the treatment period. Additionally, animals were examined for 14 days for any toxic effect. Behavioural changes and other factors such as body weight, urination, food intake, water intake, respiration, convulsion, tremor, temperature, changes in eye and skin colours, etc.^11,12

Subacute toxicity studies:

The subacute oral toxicity study of extracts of SE-oil, SE-alcohol and SE-chloro extracts of Senecio edgeworthii hook was estimated according to OECD guideline 407 on Wister rats (150-200g).

The Wistar rats were randomly allocated to three groups by sex (n = 6, M=3, F=3). The first group was provided distilled water orally (control group). Groups 2 to 7 were orally treated with (low, medium, high Doses) extracts of SE- oil 50, 200, 500mg/kg, SEC 100, 500, 1000 mg/kg and SEA 100, 500, 1000mg/kg respectively, for 28 consecutive days. All animals were supplied with feed and water ad libitum throughout the testing periods. The clinical sign was monitored daily for physiological and behavioural changes. Toxic symptoms such as toxicity and death were observed for signs of abnormalities. Bodyweight changes were recorded weekly and food consumption and water intake were observed daily. At the finish of treatment, the animals were fasted overnight but permitted administered to water ad libitum. They were then anaesthetized and blood samples were collected by retro-orbital puncture using capillary tubes with or without the anticoagulant.¹³

Haematological Analysis:

The blood samples collected with the anticoagulant were used immediately for the determination of haematological parameters such as red blood corpuscles (RBC) count, white blood corpuscles (WBC) count, haemoglobin (Hb) and platelet count were performed using a blood cell counter.¹⁴

Biochemical analysis:

The blood samples collected without the anticoagulant was centrifuged at 4,000rpm for 10 min to separate the serum. The serum was investigated for biochemical constraints such as glucose, urea, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total protein employing standard diagnostic kits.¹⁵

Necropsy:

At the finish of the protocol, the animals were anaesthetized and necropsy was performed on randomly preferred animals of each group per sex to investigate the macroscopic external features of vital organs such as the liver. The organ is cautiously detached and weighed. Organ weights were recorded in comparative terms (gram per 100g of body weight). The collected organs were fixed in 10% buffered formalin and fixed in paraffin. Histology sections (5um thick) were stained with hematoxylin and observed a light microscope.¹⁶

^{Statistical analysis:}

Statistical analysis was done by using one way ANOVA followed by Dunnett’s Comparison Test for comparison between different groups using Graph pad prism-5 software. A p < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION:

Toxicology tests in animals and humans are usually approved out on substances considered to have therapeutic potentials. This is more so essential in ensuring the superiority of herbal medicines since not all of them are as safe as they are regularly claimed. It can be injurious to take herbal medicines lacking being aware of their probable adverse effects. While the public and some health care professionals consider that herbal medicines are comparatively safe because they are ‘natural,’ there is extraordinarily little data to maintain this assumption. In addition, there are reports on the toxicities of some medicinal plants/phytoconstituents.¹⁷

Apart from the pharmacological valuable effects of Senecio edgeworthii hook thorough knowledge about the toxicology of this prominent herb is lacking. Hence, the present study was undertaken to estimate and focus on the acute and subchronic oral toxicity of Senecio edgeworthii hook in an animal model. Throughout the estimation of the toxic characteristics of medicinal plants, the purpose of LD₅₀ is usually an early step to be carried out. Facts from the acute oral toxicity study may (a) give out as the origin for classification and labelling (b) give preliminary information on the means of toxic action of a substance; (c) facilitate arrive at a dose of a new compound; (d) help in dose establish in animal studies.¹⁸

An oral acute toxicity test estimated the undesirable consequences that occur within a short time after administration of a solo dose of a test substance. For acute oral toxicity study, extracts of SE-Oil, SEA and SEC Senecio edgeworthii hook extracts were given to mice at a dose of 300mg/kg to 5000mg/kg. SEA and SEC extracts groups did not show any signs of toxicity at all doses employed. No death was observed in the treated groups, i.e., at 300mg/kg to 5000mg/kg during the study period. Therefore, the LD₅₀ of the extract could be more than 5000mg/kg. The SEA and SEC extracts may, therefore, be considered comparatively secure on acute exposure.

SE-Oil of Senecio edgeworthii hook flower hexane extract did not produce any signs of toxicity at a dose of 300mg/kg to 2000mg/kg and a dose of 2500mg/kg shows toxicity 3 out of 2 animals deaths. Therefore SE-Oil LD₅₀was found at a dose of 2500mg/kg.

An acute oral toxicity study gives a guideline to decide the doses for subacute toxic study (Low, Medium and High) which may be a more clinically applicable group after 28 days of daily treatment. On basis of acute toxicity study for subacute administration of SGE 100, 500, 1000mg/kg, SEC 50, 200, 500mg/kg and SE-Oil 50, 250, 500mg/kg extracts doses were selected of Senecio edgeworthii hook. The daily oral treatment with extracts of SEA, SEC and SE-Oil extracts for 28 days did not produce any death or hazardous sign such as piloerection, alteration in the locomotor activity and other physiological activities as compared to the control animals. Likewise, SEA 100, 500, 1000mg/kg, SEC 100, 500, 1000mg/kg and SE-Oil 50, 250mg/kg of Senecio edgeworthii hook no significant changes were recorded in food intake and body weight gain of control and treated rats result shown Table no.1 and 2. SE-Oil 500 mg/kg dose significant changes were recorded in food intake and body weight gains are decrease compare control group.

Table 1. Effect of Senecio edgeworthii extracts on food intake (gm).

Days

Groups

Control

SE-Oil (mg/kg)

SEA (mg/kg)

SEC (mg/kg)

250

500

100

500

1000

100

500

1000

± 0.25

± 0.54^ns

± 0.14^ns

± 0.32^ns

± 0.10^ns

± 0.12^ns

± 0.22^ns

± 0.34^ns

± 0.47^ns

± 0.24

± 0.32^ns

± 0.46^ns

± 0.36^ns

31 ± 0.76^ns

± 0.32^ns

± 0.41^ns

± 0.56^ns

± 0.36^ns

± 0.34^ns

± 0.44

± 0.74^ns

± 0.34^ns

± 0.54^ns

34 ± 0.44^ns

± 0.55^ns

± 0.64^ns

± 0.75^ns

± 0.56^ns

± 0.55^ns

Effect of SE-Oil, SEC and SEA extracts on food intake in rats. Data are mean SEM values (n = 6 in each group). Data were analyzed by one way ANOVA followed by Dunnett test Comparisons test. ns: Not significant *P<0.05, **P<0.01, ***P<0.001 a- compared with normal control.

Table 2. Effect of Senecio edgeworthii extracts on bodyweight

Days	Groups
	Control	SE-Oil (mg/kg)			SEA (mg/kg)			SEC (mg/kg)
	Control	50	250	500	100	500	1000	100	500	1000
7	164.33 ± 2.33	162.2 ± 3.7^ns	163.18 ± 3.5^ns	164.50 ± 1.3^ns	165.2 ± 1.7^ns	161.20 ± 2.2^ns	157.8 ± 1.3^ns	162.5 ± 1.8^ns	163.17 ± 0.6^ns	159.50 ± 3.3^ns
14	168.43 ± 1.38	167.4 ± 2.5^ns	165.17 ± 1.4^ns	165.70 ± 4.5^ns	171.4 ± 2.5^ns	167.40 ± 2.5^ns	162.5 ± 2.2^ns	168.9 ± 3.2^ns	167.7^ns ± 1.52	165.30 ± 1.8^ns
21	173.23 ± 1.39	175.2± 2.47^ns	169.25 ± 3.5^ns	164.40 ± 1.6*	175.2± 1.4^ns	172.2± 3.47^ns	169.8 ± 2.3^ns	172.2± 1.34	172.2± 2.35^ns	170.80 ± 1.5^ns

Table 3. Effect of Senecio edgeworthii extracts on haematological parameters in the subacute oral toxicity study

Parameter

Groups

Control

SE-Oil (mg/kg)

SEA (mg/kg)

SEC (mg/kg)

250

500

100

500

1000

100

500

1000

(g/dL)

12.5

± 1.2

12.42

± 1.8^ns

11.8

± 1.4^ns

11.15

± 2.1^ns

12.4

± 1.2^ns

12.8

± 1.6^ns

12.2

± 3.8^ns

12.04

± 3.2^ns

12.3

± 3.2^ns

12.16

± 1.8^ns

RBC

(10⁶/mm³)

6.5

± 2.3

6.37

± 3.6^ns

6.91

± 2.1^ns

4.72

± 1.8*

6.3

± 4.4^ns

6.1

± 4.3^ns

6.27

± 2.8^ns

6.6

± 2.3^ns

6.8

± 2.3^ns

6.7

± 2.8^ns

WBC

(10³/mm³)

7.6

± 2.32

7.3

± 3.3^ns

7.7

± 1.7^ns

8.8

± 2.5^ns

7.4

± 2.5^ns

7.2

± 1.3^ns

7.5

± 3.3^ns

7.4

±1.4^ns

7.6

±1.3^ns

6.9

± 2.3^ns

Platelets

10⁵/mm³

6.74

± 1.24

6. 12

± 3.2^ns

6.43

± 2.2^ns

6.4

± 1.2^ns

6.24

± 1.54^ns

6.7 ±1.1^ns

7. 52

± 3.2^ns

6.63

±2.1^ns

6.44

±2.2^ns

7. 2

± 3.2^ns

Effect of SE-Oil, SEC and SEA extracts on the haematological parameter in rats. Data are mean SEM values (n = 6 in each group). Data were analyzed by one way ANOVA followed by Dunnett test Comparisons test. n.s.: Not significant *P<0.05, **P<0.01, ***P<0.001 a- compared with normal control.

Table no. 4. Effect of Senecio edgeworthii extracts on biochemical parameters in subacute oral toxicity study.

Parameter

Groups

Control

SE-Oil (mg/kg)

SEA (mg/kg)

SEC (mg/kg)

250

500

100

500

1000

100

500

1000

ALT (IU/L)

36.6

± 3.3

35.6

± 2.1^ns

34.67

± 2.9^ns

42.24 ±2.5*

35.6

± 1.5^ns

36.8

± 2.7^ns

35.6

± 2.9^ns

37.5

± 3.2^ns

33.5

± 2.4^ns

37.3

± 3.5^ns

AST (IU/L)

89.97

± 7.15

91.2

± 5.6^ns

97.24

± 5.6^ns

101.9 ±1.5*

91.6

± 2.3^ns

94.99

± 5.2^ns

92.4

±2.8^ns

94.40

± 4.6^ns

93.80

± 2.7^ns

95.12

± 6.6^ns

ALP (IU/L)

101.4

± 4.2

103.2

± 4.3^ns

108.6

± 4.3^ns

109.5 ±1.2*

104.5

± 4.5^ns

102.0

± 6.5^ns

103.1

± 8.02^ns

104.5

± 7.7^ns

104.9

± 9.27^ns

101.3 ± 9.52^ns

Total protein (gm/dL)

6.9

± 1.3

6.7

± 2.5^ns

6.1

± 1.5^ns

7.9

±3.4*

6.5

± 1.2^ns

6.3

± 2.4^ns

6.8

± 1.6^ns

6.8

± 2.6^ns

6.5

± 2.6^ns

6.7

± 1.8^ns

In the hematopoietic system, individual one of the mainly vulnerable targets of toxic chemicals is a significant index of the physiological and pathological category of humans and animals. In this study, the test groups of SEA 100, 500, 1000mg/kg and SEC 100, 500, 1000mg/kg and SE-Oil 50, 250mg/kg of Senecio edgeworthii hook treaded group did not illustrate any significant variation in the haematological parameters except SE-Oil 500mg/kg there was significant (P < 0.05) decrease in RBC counts in the treated animals compared to that of the control group results shown Table no. 3.

The liver has a vital role in the metabolism of drugs or plant products. Exogenous chemicals and their metabolites might involve toxicity or cell damage on this organ. In the current histopathological inspection of the liver, treated animals of SEA 100, 500, 1000mg/kg, SEC 100, 500, 1000mg/kg and SE-Oil 50, 250mg/kg of Senecio edgeworthii hook extracts treated group showed no change in the microscopic structure of the liver result are shown fig no.1. The common architecture of the liver, the appearance of the hepatocytes, the hepatic sinusoids, portal triads, and central veins are normal as compared with controls. The result was also accompanied by the non-adverse effects of the extracts in any of the biochemical markers (such as ALT, AST and alkaline phosphatase and Total protein), which showed statistically insignificant changes compared with a control group result are shown in table no. 4. SE-Oil 500mg/kg of Senecio edgeworthii hook extract treaded group seen mild liver pathological findings characterized by abnormal hepatic configuration were observed with treated animals. SE-Oil 500mg/kg of extract treaded group significant (P<0.05) induction (ALT, AST, and alkaline phosphatase and Total protein) found in the liver and this dynamic in the respected study.

Effect of SE-Oil, SEC and SEA extracts on Biochemical parameters in rats. Data are mean SEM values (n = 6 in each group). Data were analyzed by one way ANOVA followed by Dunnett test Comparisons test. n.s.: Not significant *P<0.05, **P<0.01, ***P<0.001 a- compared with normal control.

Figure 1: Photomicrographs of rat liver tissue section under low power

A. Normal B. SE-Oil -50mg/kg C. SE-Oil -250mg/kg D.SE-Oil -500 mg/kg

E. SEC 100mg/kg F. SEC 500mg/kg G. SEC 1000mg/kg H. SEA 100 mg/kg

I.SEA 500mg/kg J. SEA 1000mg/kg

CONCLUSION:

The present result from the acute and subacute oral toxicity study Senecio edgeworthii hook SE-Alcohol and SE-chloroform extract is considered comparatively safe on acute and subacute oral exposure. The SE-Oil shows acute oral toxicity at dose 2500mg/kg and in subacute toxicity study, higher dose 500mg/kg shows by abnormal hepatic configuration were observed with treated animals.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 20.01.2022 Modified on 14.04.2022

Research J. Pharm. and Tech 2023; 16(4):1901-1906.

DOI: 10.52711/0974-360X.2023.00312