Asep Arifin Senjaya^1*, Ni Nyoman Dewi Supariani², and Ni Made Sirat³

¹Midwifery Departement - Polytechnic of Health Denpasar, Bali, Indonesia

²Dental Hygiene Department - Polytechnic of Health Denpasar, Bali, Indonesia

Corresponding Author E-mail:  aseparifinsenjaya@gmail.com

Abstract

The efficacy of traditional medicines is related to the complexity of the chemical properties of the drugs. Cosmos caudatus Kunth is a traditional medicinal plant with therapeutic properties. This study aimed to determine the chemical compounds contained in C. caudatus Kunth leaves and their toxicity. Toxicity tests were conducted on 24 male mice (Mus musculus) divided into one control group consisting of aguadest and five treatment groups consisting of C. caudatus Kunth ethanol extract at doses of 125mg/kg bw, 250mg/kg bw, 500mg/kg bw, 1g/kg bw, and 2g/kg bw. Phytochemical analysis showed that the ethanol extract of C. caudatus Kunth leaves contained alkaloids, tannins, phenols, flavonoids, and saponins. The highest levels of alkaloids, tannins, phenols, and flavonoids were found in the ethanol extract of C. caudatus Kunth leaves fractionated with aqua, while the highest level of saponins was found in the aqua fractionation extract. The highest test dose of 2g/kg bw did not cause poisoning or death in mice. There were no significant differences in liver weight (p=0.14), kidney weight (p=0.44), or creatinine (p=0.21) between the control and treatment groups. Histopathological examination of the liver showed severe hydropic degeneration that was not significantly different between the control and treatment groups. Renal histopathology showed significant differences between the control and treatment groups. Conclusion: ethanol extract of C. caudatus Kunth leaves contains alkaloids, tannins, phenols, flavonoids and saponins. A dose of 2 g/kg bw of ethanol extract of C. caudatus Kunth leaves was not toxic to mice, but most of the mice's livers experienced severe damage.

Keywords

Phytochemicals; toxicity; extract of C. caudatus leaves; invivo

Copy the following to cite this article: Senjaya A. A, Supariani N. N. D, Sirat N. M. Phytochemicals and Toxicity of the Extract from Cosmos caudatus Leaves. Biomed Pharmacol J 2024;17(3).

Copy the following to cite this URL: Senjaya A. A, Supariani N. N. D, Sirat N. M. Phytochemicals and Toxicity of the Extract from Cosmos caudatus Leaves. Biomed Pharmacol J 2024;17(3). Available from: https://bit.ly/4cxCjZ9

Introduction

Around 80% of the world’s population is estimated to use traditional medicine¹. Most of the Indonesian population, especially in rural areas, use traditional herbal medicines². Its efficacy is related to the complexity of the chemical nature of drugs – traditional medicines offer many advantages in terms of efficiency and molecular target selectivity³.

Phytochemical tests of medicinal plants continue to be developed to develop new medicinal ingredients. Qualitative determinations in phytochemical screening provide information on the presence of certain compounds or groups of compounds, and quantitatively distinguish which are the main components and which are additional components in the mixture. Isolation of secondary metabolite compounds is carried out through an extraction process using organic solvents with increasing polarity sequentially.⁴ The polarity of the type of solvent used in extraction must be the same or very close to the polarity of the active ingredient being extracted so that the extraction runs efficiently because according to the principle like dissolves like not all compounds will dissolve in a liquid solvent.⁵

C.caudatus Kunth originates from Mexico to South Tropical America. This plant is an annual and grows primarily in seasonally dry tropical biomes. C.caudatus in Malaysia is called ulam raja⁶ and kenikir in Indonesia.⁷ one of the traditional medicinal plants with medicinal properties. Its leaves are often consumed by Indonesians. C. caudatus leaf extract shows the presence of active compounds such as flavonoids, saponins, alkaloids, tannins, and polyphenols.⁸  Traditionally used as antihypertensive, antidiabetic, antioxidant, antiosteoporosis, antifungal, and antibacterial⁹. C. caudatus rich in flavonoid glycosides exhibits skin anti-aging effects through inhibiting collagenase, MMP-1 and MMP-3 activities, possibly via the NF-κB pathway.¹⁰ Although traditional medicine has been used for a long time, it is very important to know its potential toxicity.¹¹ The medicinal-activity plants should have low toxicity because of their long-term use in humans.¹²

Acute toxicity testing is intended to obtain information on poisoning symptoms, causes of death, sequence of death processes and lethal dose range for test animals by a substance.¹³  Observations on experimental animals are carried out within 24 hours after giving the test material to the emergence of symptoms of poisonin such as body weight, drowsiness, lacrimation, nasal bleeding, paralysis, piloerection, salivation, skin, utilization of food, water and death and changes in the function of vital organs of the experimental animals. Acute toxicity testing (LD50) can be seen from changes in the structure and function of vital organs such as the liver and kidneys.¹⁴  .  The purpose of this study is to determine the chemical compounds contained in C. caudatus leaves as well as their toxicity.

Material and Method

Plant material

The leaves of C. caudatus were dried under room temperature with further drying using an oven and ground to fine powder using an electric grinder. 200 g of C. caudatus leaf simplicia was macerated with 2000 ml of 96% ethanol for 72 hours at room temperature. The filtrate is obtained by filtering with Whatman No. 1 filter paper. The filtrate obtained was then evaporated at a temperature of 40⁰C, to obtain a crude extract in paste form, assumed to be a concentration of 100%.¹⁵  The crude ethanol extract is then fractionated for polar compounds with aqua, semi-polar with ethyl acetate, and non-polar compounds with hexane.¹⁶  

Phytochemical compound test

Qualitative testing of C. caudatus extract was carried out according to standard methods to detect the main phytochemicals such as alkaloids, flavonoids, saponins, sterols, sugars, phenols etc. present in the extract.The fractionation results were then examined for the content of alkaloids, tannins, saponins, flavonoids and phenols, steroids and terpenoids.¹⁷ Quantitatif phytochemical test have been carried out on C. caudatus leaf extracts using the UV Visible Spectrophotometry method.

Quantification of total content of alkaloid compounds

Quantitative analysis of alkaloids was carried out using the spectrophotometry method. 1ml of the sample extract was mixed with 1 ml of 0.025 FeCl3 in 0.5M HCL, and then add 1 ml of phenanthroline. The resultant mixture formed was incubated for 30 minutes in a water bath maintained at  70oC. Then, measure the absorbance of the sample using UV-Visible spectrophotometer with a wavelength of 510 nm. The alkaloid content of samples is expressed in mg/100g sample weight.¹⁸

Quantification of total content of tannin compounds

A total of 0.01 g of extract was diluted in 5 ml of citrate phosphate buffer. The diluted sample was pipetted to 0.25 ml then 0.25 ml of Folin-Denis reagent was added, then vortexed and 2 ml of 5% Na2CO3 was added. The solution was vortexed and then incubated for 30 minutes. Absorbance was read at a wavelength of 725 nm using a UV-Visible spectrophotometer. The reading results were compared with a standard curve using tannic acid. Total tannins in samples were expressed as tannic acid equivalents in mg TAE/g extract.¹⁹

Quantification of total content of saponin compounds

The mixture containing 250 µL of vanillin reagent, 50 µL of extract and 2.5 mL of 72% sulfuric acid was mixed and incubated at 60 °C for 10 min. At the end of the incubation, the absorbance of the solution cooled in an ice bath was read at a wavelength of 544 nm. Saponin content was expressed as mg diosgenin equivalent (DE)/g.²⁰

Quantification of total content of phenolic compounds

Folin-Ciocalteu method was employed for the quantification of total phenolic content.  A total of 0.01 g of diluted extract into 5 ml of citrate phosphate buffer according to treatment. A sample of 0.1 ml was pipetted and 0.3 ml of 70% ethanol was added. After that, 0.4 ml of Folinciaocalteau was added and then incubated for 6 minutes. After the incubation process, 4.2 ml of 5% Na2CO3 was added, then vortexed and incubated for 90 minutes. Absorbance was read at a wavelength of 760 nm with using UV- Visible spectrophotometer. The content of total phenolic compound was denoted as mg of GAE/g of extract.²¹

Quantification of total content of flavonoid compounds

Determination of total flavonoids using a spectrophotometer with the AlCl3 method refers to (Singh et al., 2012). A total of 0.01 g of extract was diluted in 5 ml of phosphate citrate buffer according to the treatment. A total of 1 ml of sample was mixed with 4 ml of distilled water and 0.3 ml of NaNO2 solution (10%) was added. After that, it was incubated for 5 minutes and 0.3 ml of AlCl3 solution (10%) and 2 ml of NaOH solution (1%) were added, then immediately tested with a spectrophotometer at a wavelength of 510 nm. Flavonoid concentrations in the test samples were calculated C x V x FP W 37 from calibration standards prepared using quercetin standards and expressed as quercetin equivalents in mg QE/g extract.²²

Animal preparation

24 male mice (Mus musculus) of the balb/c strain, aged 2.5 to 3 months and weighing between 25 and 30 grams, were used as test animals. Acute toxicity testing of traditional medicine should be performed on at least one rodent species, namely mice or rats.²³ Male mice are not affected by the estrus cycle.²⁴ The mice used were acclimatized for 10 days at room temperature. The mice were placed in a plastic cage. Every three days, the mouse cage was cleaned and the bedding was changed. The mice were fed chicken pellets once a day in the morning and given water ad libitum.

Experimental groups

The recommended dose is a minimum of four levels.¹³  Referring to Herlina et al (2021),  the LD50 test dose for this study: 125mg, 250mg, 500mg, 1g and 2g/kg BW of mice.²⁵ 24 experimental animals were randomly divided into one control group, namely distilled water and five treatment groups, namely C. caudatus ethanol extract 125mg/kg bw, 250mg/kg bw, 500mg/kg bw, 1g/kg bw and 2g/kg bw. Before treatment, mice were fasted for 10 hours, to avoid food factors. Each experimental animal was given 1 cc of the extract solution according to the dose. The extract is given in a single dose orally using a gastric probe. Observation of acute toxic effects was carried out within 1 x 24 hours.²⁴  After 1 x 24 hour observation, the mice were anesthetized with a mixture of ketamine + zylazine intramuscularly in the thigh muscle as much as 0.02 cc. Blood is taken using capillary hematocrit, in the retro orbital area as much as 0.8cc – 1cc. Dead mice were dissected to remove the liver and kidneys, the weighed. The liver and kidneys of mice were washed with 0.9% NaCl. The liver and kidneys were fixed for 1×24 hours in 10% buffered formalin. Next, trimming, tissue dehydration, clearing, impregnation, embedding, cassette is removed from the tissue processor and placed in a paraffin bath, clean the brass blocks, arrange them neatly. Take the specimen from the embedding cassette with tweezers, put it on a brass block, pour the liquid paraffin on the embedding machine into the block, give a marker number, cut the block to a thickness of 4 microns. The preparation was floated in a water bath. The preparations were placed in an incubator for one night. Stain the preparations with Harris-Haematoxyllin-eosin (HE) dye. Histopathological examination of the liver and kidneys was carried out at 400X magnification.²⁶

Statistical analysis

The data were statistically tested with One Way Anova and Kruskal Wallis  in SPSS statistic. Statistical test results are significant if p <0.05.

Results and Discussion

Extracts of C. caudatus leaves were fractionated into three categories: water, ethyl acetate, and hexane. All three categories were found to contain alkaloids, tannins, saponins, flavonoids, and phenols, but none of them contained steroids and terpenoids. This suggests that the compounds present in the extract are polar, semi-polar, and non-polar. Further studies by Vikneswari Perumal et al. in 2014 also supported these findings, as they discovered that C. caudatus leaves contain active compounds such as flavonoids, saponins, alkaloids, tannins, and polyphenols.⁹ However, Muhamad Dea Firdaus et al. in 2021 found that the aqua extract of C. caudatus leaves only contained alkaloids and tannins, while the 50% ethanol extract contained alkaloids, flavonoids and tannins, and the 96% ethanol extract contained alkaloids, tannins, saponins, and terpenoids. This could be due to variations in harvest time, weather conditions, and environmental temperature.²⁷

Table 1:  Qualitative Phytochemical Test of  Ethanol Extract C. caudatus Leaves

	Test	Aqua			Ethanol
		Aqua	Ethyl Acetate	Hexane	Aqua	Ethyl Acetate	Hexane
Alkaloid	Drangendroff	+	+	+	+	+	+
Tannin	FeCl3	+	+	+	+	+	+
Saponin	Foam	+	+	+	+	+	+
Flavonoid	HCl	+	+	+	+	+	+
Fenol	Folin-Ciocalteau	+	+	+	+	+	+
Steroid	Chloroform, acetic acid, concentrated sulfuric acid	–	–	–	+	+	–
Terpenoid	Chloroform, acetic acid concentrated sulfuric acid	–	–	–	–	–	–

 

The results of the Kruskal-Wallis test indicate a significant difference in the mean levels of alkaloids, tannins, saponins, phenols, and flavonoids in various plant extracts. The obtained p-value is 0.000. The highest levels of alkaloids, tannins, phenols, and flavonoids were found in the ethanol extract of C. caudatus leaves with water, while the highest level of saponin was found in the water extract fractioned with water. According to Stevens GW et al.²⁸, ethanol is a versatile solvent and is excellent for preliminary extraction, extracting bioactive compounds faster. Ethanol and water are polar solvents. The research results show that 96% ethanol solvent is better than water for extracting polar compounds such as alkaloids, tannins, phenols, and flavonoids.

Table 2: Quantitative Phytochemical Means of C. caudatus Leaf Extracts

Extract	Alkaloid (mg/100g)	Tannin (mgTAE/g)	Saponin (mgDE/g)	Phenol (mgGAE/g)	Flavonoid (mgQE/g)
Aqua fractionated aqua	14.837 ± 0.985	626.885 ± 9.273	19.615 ± 7.117	615.172 ± 2.438	252.031 ± 2.209
Aqua fractionated ethyl acetate	13.568 ± 0.150	9.410 ± 0.231	11.957 ±1 4.959	24.678 ± 0.162	13.484 ± 0.441
Aqua fractionated hexane	6.036±0.892	2.114 ± 2.434	2.221 ± 1.196	17.552 ± 0.487	3.797 ± 0.000
Ethanol fractionated aqua	18.881 ± 2.457	1162.798 ±13.772	18.430 ± 6.644	1073.973 ±25.751	1019.957 ± 2.187
Ethanol fractionated ethyl acetate	13.381 ± 1.074	95.991 ± 2.066	9.700 ± 2.256	71.549 ± 1.448	34.390 ± 0.000
Ethanol fractionated heksana	6.914 ± 0.696	31.634 ± 0.306	5.026 ± 4.635	26.693 ±0 .710	10.285 ± 0.965
p value	0.000	0.000	0.000	0.000	0.000

 

The results of the LD50 test showed that all mice moved agilely, behaved in kissing/smelling each other, showed no signs of poisoning and no deaths were found. According to Gil, M.I., et al. (2002), the absence of death in experimental animals means that the LD₅₀ value does not need to be determined.¹⁶  The most severe toxicity categories (LD₅₀ ≤ 0.005 g/kg bw), relatively low toxicity (LD50 > 2−5 g/kg bw), very low toxicity (LD50 > 5 g/kg bw).²⁹  The highest dose tested was 2 g /kg BW so a larger dose needs to be tested on mice to determine the LD50.            

The weight of the liver and kidneys does not show a tendency to increase or decrease with the dose given. The one-way Anova test results for the difference in the weight of the mouse liver had a p-value of 0.14 (> 0.05). This means that there is no significant difference in the weight of the mouse liver between treatments and control groups. The one-way Anova test results for the difference in the weight of the mouse kidney had a p-value of 0.44 (> 0.05). This means that there is no significant difference in the weight of the mouse kidney between treatment and control groups. Most liver cells are composed of parenchymal cells such as hepatocytes, as well as other non-parenchymal cells present in sufficient numbers.. Changes in liver and other organ weights may be temporary. According Michael et al., in toxicity studies, organ weight changes are sensitive indicators of toxicity, effects on enzymes, physiologic disturbances and target organ injury. Teo et al. stated that an increase in organ weight suggests the occurrence of hypertrophy while a decrease suggests necrosis in the target organ.³⁰ The liver has the extraordinary ability to regenerate to its original size after injury. The stability of liver function and size is essential for whole body homeostasis. ^31,32  

Table 3: Liver and Kidney Weight Mice After  LD₅₀

Groups	Liver weight (gr)	Kidney weight (gr)
Control	1.257 ± 0.155	0.682 ± 0.557
P1	1.540 ± 0.144	0.235 ± 0.034
P2	1.295 ± 0.124	0.185 ± 0.031
P3	1.542 ± 0.227	0.232 ± 0.037
P4	1.637 ± 0.372	0.165 ± 0.036
P5	1.470 ± 0.208	0.215 ± 0.046
p value	0.14	0.44

 

One way to determine kidney function is by determining serum creatinine and urine creatinine clearance which is commonly used and highly sensitive^16,31 The one way Anova test for mean creatinine differences yielded a p-value of 0.21 (> 0.05) indicating  no significant difference in serum creatinine levels between treatment groups and the control group. Normal creatinine levels in mice range from 0.2 – 0.8 mg/dl.³³ Ethanol extract of C. caudatus leaves had no effect on the kidney function of mice, as indicated by the normal serum creatinine levels in all mice. Creatine levels can increase if there is renal or glomerular dysfunction.³⁴ Since this study used mice, the amount of serum obtained was limited. If the study wereconducted using rats,  more serum could be obtained, which could be used to examine liver function through AST and ALT tests.

Table 4: Creatinine Serum Means (mg/dl)

Groups	Creatinine Serum
P1	0.50 ± 0 .000
P2	0.55 ±  0.057
P3	0.55 ± 0.057
P4	0.50 ± 0.000
P5	0.55 ± 0.057
Control	0.50 ± 0.000
p value	0.21

The Kruskal Wallis Liver Test results obtained a value of p = 0.331 (> 0.05), indicating that  there was no significant difference in liver damage in mice between the treatment and control groups. The histopathological picture of all treatment and control groups showed that hepatocyte cells experienced a severe degree of hydrophic degeneration. The liver is an organ that has the potential to experience damage due to various chemicals and the environment because of its function in the metabolic process and as a detoxification center for toxic substances in the body. Administration of toxic compounds can cause changes such as hemorrhage, congestion, degeneration and necrosis.²⁴

Degeneration is divided into two types, namely parenchymatous degeneration and hydropic degeneration. Hydropic degeneration, often called ballooning degeneration, is degeneration that occurs in the liver which has the characteristics of liver cells swelling up to twice normal, and is reversible²⁸. The liver plays a central role in nutrient metabolism, glucose and lipid synthesis, and detoxification of drugs and xenobiotics.³⁵ The liver is often the target organ that is injured by chemicals. Many compounds can cause liver cell damage. Oxidative stress results in excess pro-oxidants causing damage to cells, often resulting in cell death.¹⁴       This research shows that after the LD₅₀ test , the degree of damage to the liver is more severe than the kidneys in mice.

On the other hand, the test for differences in kidney damage obtained a sig. value: 0.002 (< 0.05), meaning there was a significant difference in kidney damage in mice between the treatment group and the control group. The kidney histopathology seen in the control group was all normal, whereas in the treatment group there was multifocal damage. Kidney is an organ of the body that is susceptible to the influence of chemical substances because the kidneys work in the rest of the metabolic products of the blood so that the possibility of pathological changes is very high.³⁶  The susceptibility of the kidney to toxic injury is related to the complexity of renal anatomy and physiology. Nephrotoxicity can be a serious complication of drug therapy or chemical exposure.¹⁴ Glomerular function can be observed by measuring urea and creatinine levels, but histopathological testing remains the gold standard in identifying nephrotoxicity.³⁷

Many plant families contain potentially toxic alkaloids.³⁸ Secondary metabolite compounds in plants include alkaloids, flavonoids, saponins, tannins, steroids, triterpenoids, and others which are basically toxic to plants and animals. In some plants secondary metabolite compounds are used to defend themselves from enemies, but at certain doses they can be used as medicine.³⁹  Some agents can cause hypersensitivity or allergic reaction which manifests as inflammatory infiltrate in the interstitial and tubular compartment. Several plant toxicity studies show that increasing doses of herbal medicines causes cell degeneration activity which worsens the condition of kidney function. Likewise, the results of this study show that kidney damage increases with increasing dose of the test substance.⁴⁰Possible damage to the liver and kidneys of mice after the LD50 test is due to secondary metabolites contained in the ethanol extract of C. caudatus leaves.

Table 5: Histopathology of Mouse Liver

Mouse Number	Groups						p value
	Control	P1	P2	P3	P4	P5
1	3	3	3	0	3	3	0.331
2	3	0	3	3	3	3
3	0	3	3	3	3	3
4	0	3	3	3	3	3

Table 6: Histopathology of Mouse Kidney

Mouse Number	Groups						p value
	Control	P1	P2	P3	P4	P5
1	0	0	0	0	2	2	0.002
2	0	0	1	2	2	2
3	0	0	1	1	2	2
4	0	1	1	1	2	2

Description⁴¹ :

Score 0: normal or no damage

Score 1: there is focal (mild) damage, damage in one place

Score 2: there is multifocal (moderate) damage, damage in several places

Score 3: there is diffuse (severe) damage, damage all over the place

Damage in the form of hydrophic degeneration in the liver and glomerular shrinkage in the kidneys

Conclusion

The ethanol extract of C. caudatus leaves contains alkaloids, tannins, phenols, flavonoids and saponins. A dose of 2g/kg BW extract is not toxic to mice. After being given ethanol extract of C. caudatus leaves, most of the mice’s livers experienced severe damage and most of the mice’s kidneys were in normal condition. It is recommended to use larger experimental animals such as white mice, so that the amount of serum obtained is greater, so that AST and ALT can be checked as parameters of liver function.

Acknowledgement

We are very grateful to all the staff of the Polytechnic of Health Denpasar, Bali, Indonesia for their support in carrying out research until it is ready to be published.

Conflict of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper. We certify that the submission is original work and is not under review at any other publication.

Funding source

This study was supported by the Polytechnic of Health Denpasar, Board for Development and Empowerment Human Resources of Health – The Ministry of Health Republic Indonesia, grant number HK.02.03/WD.1/3942/2023.

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