Phytochemical Test and Acute Safety Evaluation of Oral Purple Leaves ( Graptophyllum Pictum L. Griff) Extract in Rats

Background: Purple leaves ( Graptophyllum pictum (L) Griff ) is a native plant from Indonesia (Polynesia) which is empirically common used to treat hemorrhoids, diabetes


INTRODUCTION
Purple leaves (Graptophyllum pictum (L) Griff) is a native plant from Indonesia (Polynesia) which is empirically common used to treat hemorrhoids, diabetes, and many other diseases. 1, 2 The nano technology of this product also have been widely developed. [3][4][5] Despite its massive development globally, there are few studies have written about the toxicity of this herbal medicine. The aim of this study is to describe the acute toxicity of this herbal medicine, as the basic ground of the further safe development of this medicine.

MATERIALS AND METHODS
The process of making purple leaf extract Fresh purple leaves (Graptophyllum pictum (L)griff) harvested from the Herbal Plants Resource Garden of Indonesian Medicinal and Aromatic Crops Research Institute (IMACRI), Bogor, Indonesia. They were washed with running water, sorted wet to sort the fresh leaves and then drained. Fresh purple leaves were dried using an oven (50°C) and then the moisture content was measured using a moisture balance device. The dried purple leaves were blended and sieved using a mesh sieve no.40. Each purple leaf dry powder (2 kg) was extracted with 15 L of 70% ethanol and 15 L of hexane by maceration method for 3 days at room temperature and then filtered to obtain macerate liquid. Each process was repeated 2 times and each macerate was concentrated using a rotary evaporator and followed by freeze-drying to produce 70% ethanol extract and purple leaf hexane extract.

Phytochemical qualitative analysis
The ethanol extract of G. pictum was tested using standard procedures with specific reagents to detect the presence of secondary metabolites such as alkaloids, saponins, tannins, phenolics, flavonoids, triterpenoids, steroids, and glycoside.

Alkaloid test:
The presence of alkaloids was tested using three reagents namely, Dragendorff's reagent, Wagner's reagent, and Mayer's reagent. Each test tube was filled with 500 μL of extract solution. Subsequently, a few drops of reagent were added into the tube. A reddish-brown precipitate indicated the presence of alkaloids. 6 Saponins: Few drops of Na2HCO 3 were added to the extract solution (500 μL) and shaken for 5 minutes. The presence of saponin was indicated by the formation of froth or lather. 6 Tannins: A few drops of 5% w/v ferric chloride solution were added to 1-2 mL of the extract. A greenish color indicated the presence of gallotannins, while brown color indicated the presence of pseudotannins. 6 Triterpenoids: The dry crude plant extract (5 mg) was dissolved in chloroform (2 mL) and then acetic anhydride (1 mL) was added to it. Concentrated sulphuric acid (1 mL) was added to the solution.
Formation of reddish violet colour shows the presence of triterpenoids.

Steroids:
Chloroform was added to the extract solution (500 μL) followed by conc. H2SO4 added slowly through the sides of the test tube. The lower sulphuric acid fraction turned brownish yellow and the upper layer turned reddish orange which indicated presence of steroids.
Glycoside: Few drops of aqueous NaOH were added to the extracts (500 μL). Yellow colored solution indicated presence of glycosides.

Phenolics:
To the test solution (500 μL), a few drops of FeCl3 were added. Presence of phenols was indicated by formation of a blue or blue-green colored solution (500 μL). 7 Flavonoids: A few drops of NaOH solution was added to the extract solution (500 μL) followed by diluted HCl. The solution turned yellow and then colorless, indicating the presence of flavonoids. 6

Quantitative analysis of chemical constituents
Total saponins: Five µL extracts (1 mg/ml) were separately applied (samples and standard) to the plate. The mobile phase consisted of hexane-ethyl acetate-methanol (4:4:1) and 20 ml of mobile phase was used per chromatography. Linear ascending development was carried out glass chamber saturated with the mobile phase. The developed plate was scanned at 301 nm using CAMAG TLC scanner equipped with win-CATS-software.
Total flavonoids: Total flavonoid content was estimated by the method of Sathish Kumar et al. using Quercetin as reference compound. 8 A volume of 10 mL of extract was added to 0.5 ml of sodium acetate 0.5% solution, 2 ml AlCL3 (2%), added acetic acid up to 50 ml, and kept at room temperature for 25 minutes. The absorbance for the extract and the standard solutions was measured with a UV/Visible spectrophotometer at 425 nm. The standard solution was prepared using a quercetin solution (0-1 mg/mL) to generate a standard curve (r 2 = 0.999). The amount of flavonoids in the extract was presented as per cent quercetin.

GC-MS analysis of extracts:
GC-MS analysis was employed for the identification of chemical constituents in PLE and PLH. An Agilent 7890 series GC coupled with a 5975 mass selective detector was equipped with an Agilent HP ultra 2 capillary columns (5%-phenyl)methylpolysiloxane phase, 30 m x 0.20 mm I.D. with 0.11 µm film thickness) as stationary phase for compound separation. Helium gas at a constant flow rate of 1.2 mL/min. was used as carrier gas. Five µL of each extract was subjected to GC-MS analysis in 8:1 split mode with GC inlet temperature of 250 °C. The GC oven temperature program was started at 80 °C. Subsequently, it was gradually increased at the rate of 3°C/min until reaching 150 °C. The oven temperature was then maintained at 150°C for another 1 min. Finally, the temperature gradually increased at the rate of 20°C/min to 280°C/min and hold for 26 minutes. MS detection was carried out in electron impact (EI) mode with ionization energy of 70 eV. The temperature of transfer line, MS source, and MS quadrupole were kept constant at 280, 230, and 140°C, respectively. A full scan acquisition mode was performed for MS analysis in the mass range from 30 to 600 m/z. The identification of constituents was accomplished by matching their mass spectral data with those in the Chemstation Data System.
Acute toxicity study of purple leaves extract in rats 36 male Sprague-Dawley (SD) rats, aged 6 to 8 weeks, were used in the investigation. Every day, the rats have free access to food and water. During the experiment, the animal room's temperature was roughly 22+3 °C, relative humidity was around 80%, and there was a 12-hour light-dark cycle. Every day, all rats have free access to food and water from the faucet.
The three ethanol extract groups and the three hexane extract groups were split up into seven groups of rats at random. Procedures set by the Organization for Economic Co-operation and Development are followed for conducting toxicology tests (OECD). The doses of the test preparations given were 50, 300 and 2000 mg/kg of the test extract given to each test animal in the treatment group orally. The extract was mixed with water and amount of 20 cc/kg was fed by oral tube thrice daily. The rat fasted in the following 3 hours after administration of extract. Test animals were observed intensively for indications of toxic effects during the first 6 hours, 12 hours, 24 hours after administration of the extract and then observed every day for 14 days. Rats were weighed and observed every day for death, behavioral patterns, physical changes, pain, injuries and signs of illness. On day 14th, blood was withdrawn via tail veins, then, rats were sacrificed, and vital organs such as liver, kidney, heart, lungs and spleen were taken and examined for lesions. All organs are weighed individually. The liver and kidneys were then used for histopathology. The procedure of necropsy and sampling of the liver and kidney was as described in Fiette. 9 Blood samples were used to assess the hematological profiles (including the number of white blood cells, red blood cells, hemoglobin, hematocrit, MCH, MCHC, and platelets) and blood chemistry (including the levels of glucose, urea, creatinine, total protein, ALT, and AST) to assess the health of the liver and kidneys.

Statistical analysis
The results were displayed as the mean standard deviation (SD). The homogeneity of the data was determined using Saphiro Wilk. The paired student t test was used to compare pre-and post-treatment body weight, whereas ANOVA was used to examine hematological data. P values less than 0.05 were considered statistically significant. The statistical analysis was evaluated by Jamovi 2.2.2 and displayed in a table.

RESULTS
The purpose of the qualitative phytochemistry is to determine at an early stage which bioactive compounds are suitable for simplicia and its extracts, as well as to establish the plant's potential drug activity. This study demonstrated that Graphtophyllum pictum L. Griff extracts etanol and hexane contained metabolite seconder such as alkaloid, flavonoid, fenol, saponin, and tannin (Table 1).

Acute toxicity of extract ethanol and hexane in rats
Acute toxicity of ethanol and hexane extract were assessed following a single dose administered by gavage at a dose of 50, 2000 and 3000 g/ kg bw. All rats were survived for 14 days and all animals gained more weight and appeared active. The mean body weight of rats increased from 150 ± 2.22 g to 161 ± 6.68 g during the 14 days. The difference of body weight between before and at 14 days among groups were significantly different with P value <0.01.
The observation from macroscopic and gross pathology performed at necropsied yielded no visible lesions in any animals. Therefore, no evidence of acute toxicity of ethanol and hexane extract of G. pictum in rats was found. The oral LD 50 values for male rats must be greater than 3000 g/kgBW.
All hematological parameters measured (haemoglobin, haematocryte, leucocyte) were not significantly different. The thrombocyte level was significantly different amongst group (P value 0.033). The results of Makkiyah F, et       urea, creatinine, SGOT and SGPT were within normal range and with not significantly different between control and treatment groups ( Table 2).

DISCUSSION
Natural products, including minerals, plants and animals, have been used as drugs since ancient times. There are so many plants used for traditional medicine, such as in Ayurvedic Medicine, Chinese Traditional Medicine, Unani Medicine, etc., which probably possess therapeutic effects and need to be proven for its efficacy and safety to fulfil the modern standards of therapeutic agents. 10 The increased use of herbal plants as remedy for various diseases is gaining massive attention due to their lower allergenicity. 11 However, the potential toxicity in many of bioactive substances must be investigated to claim the safety of the herbal.
In this study, phytochemical screening qualitatively showed the presence of flavonoids, tannins, saponins, alkaloids and phenolics in both ethanol and hexane extracts of G. pictum. Recent studies on the chemistry of G. pictum have shown the occurrence of phenol, flavonoids, tannin, alkaloid, saponin, terpenoid and steroid. 1 Estimation of amount of phytochemical demonstrated that PLH (Purple Leave Hexane) contained more flavonoids, saponin, and tannin than PLE. The DPPH method showed that PLE (Purple Leave Ethanol) and PLH possessed low antioxidant activity with IC 50 greater than 500 μg/mL. Interestingly, in contrast with these findings, Rustini & Ariati 12 found that PLE possessed high antioxidant activity compared to other extracts.
The use of G. pictum preparation as a treatment of disease is very common. In Indonesia, it has been popularly used as treatment of haemorrhoid. Some of these usages have been studied in vitro and in vivo. Recently many commercial products in form of jamu, powder and capsule containing G. pictum extract have become available in the market, especially in Indonesia.
The results of acute toxicity study indicated that PLE and PLH (Purple Leave Hexane) did not cause visible signs of toxicity or mortality. The body weight changes have been observed for 14 days of experiment. However, the results showed that PLE and PLH did not significantly affect body weight as compared to corresponding baseline values. These results suggest that the extracts did not disturb rat growth; this is with the agreement to the toxicity classification reported by Loomis and Hayes. 13 The macroscopic examination of vital organs showed no abnormality. The histological evaluation did not reveal any pathology after PLE and PLH treatment. Furthermore, haematological biochemical values were found normal in PLE and PLH treated groups compared to normal group. This result indicated the evidence of safety at the tested doses when administered orally. The safety of treatment was also confirmed by the absence of behavioural changes and absence of difference in feed consumption between the treated and normal control groups. Hence, the results clearly showed that oral administration of PLE and PLH are safe at the evaluated doses (50, 2000 and 3000 mg/Kg) in Sprague Dawley rats. In this context, the data presented in this report would further in utilizing the traditional medicinal and health benefit attributes of G. pictum, which is finding increasing use in several herbal products that are currently being marketed in Indonesia.

CONCLUSION
The current study showed that hexane extract contains more valuable components for medical treatment purposes. The acute toxicity on 50, 2000,3000 kg/BW oral ethanol and hexane extract of Graptophyllum pictum showed no significant influence on hematological blood parameters of rats. It is safe to administered orally ethanol or hexane extract of G. pictum below 3000mg/kgBW rats.

ETHICAL APPROVAL AND CONSENT TO PAR-TICIPATE
Ethical approval was derived from UPN Veteran Jakarta commission ethics, Number: 2770/IX/2020/KEPK.

CONSENT FOR PUBLICATION
This study does not relate to patients, there is no need consent for publication.

AVAILABILITY OF DATA AND MATERIALS
Authors are ready to give the data when it is needed.