Determination of Marker Compounds of Senna alata from different location in East Kalimantan, Indonesia

Senna alata is a plant used for medical purposes, and its leaves have an extended history of use as a traditional herbal medicine in Indonesia. S. alata is known to contain some components of biologically active compounds and also secondary metabolites. In fact, S. alata can grow well in various locations in Indonesia, location differences can lead to differences in compound content due to differences in environmental conditions such as soil, rainfall, light intensity, and humidity. Therefore, this study aimed to analyze marker compounds of S. alata plants origin from three different location in East Kalimantan, i.e Samarinda, Samboja, and Berau. This research was conducted to estimate the compounds contained in extracts by using GC-MS analysis, and to discover relationships between different variables by using Principal Component Analysis (PCA). There are variances in the yield of secondary metabolites according on where Senna alata is grown, specifically in the riverside Nyapa Indah region and the plains of Samarinda and Samboja. Based on GC-MS test results, Phytol was the main compound in S.alata in two areas, i.e. Samarinda and Samboja. Meanwhile, in Berau showed that 1,2-Benzenedicarboxylic acid, and mono (2-ethylhexyl) ester were the main components. However, S. alata leaf extracts could be used as a good quality raw material for pharmaceutical industries, such as a laxative agent.


Introduction
Senna alata or known as urokep is known to contain components of biologically active compounds and secondary metabolites.Secondary metabolites of urokep are widely described by researchers for their various benefits, such as phenols (rhine, kaempferol, chrysophanol, glycosides, and aloe-emodin), anthraquinones (alatonal and allatinone), fatty acids (linoleic, oleic, and palmitic acids), steroids and terpenoids (campesterol, stigmasterol, and sitosterol).[1,2].These secondary metabolites exhibit various biological activities, one of which is considered a slimming agent [1,3].In some of traditionally used, Senna alata is a shrub to treat infections to parasitic skin diseases and infection against ringworms [4].
Senna alata can be grown in various locations, such as France [5] Brazil, Ghana, Australia, India, Egypt, Sri Lanka, Somalia, and through Africa [6].It is a beautiful flowering plant that is indigenous to the Amazon Rainforest.[38].Similar to other species of Senna, it prefers to flourish in the wet and warm climates found across Africa [7], Brazil, Asia, India, West Indices, Australia, Mexico, South America, Hawaii, Polynesia, Melanesia, and the Caribbean Islands [2,5].In the Thailand, Philippines, and Indonesia, this shrub has a broad distribution and is utilized for pharmacological purposes [8].
Differences in location can result in variations in the composition of compounds due to variations in environmental variables such as soil, rainfall, the intensity of light, and humidity.According to the findings of a few pieces of research, the phytochemical make-up and the levels of biological activity of plants can change greatly based not only on where they are grown but also on the ecological conditions that are present [9,10].However, each species has marker compounds [11].Therefore, this study aimed to analyze marker compounds in urokep plants for its bioactive compounds with pharmacologically important therapeutic.

Sample Collection
S. alata l e a v e s were collected from three locations i.e., Samarinda, Samboja, and NyapaIndah, East Kalimantan, Indonesia, in June 2021.

Maceration
During this step, 300 grams of urokep leaf are put into a container with a lid, along with 500 milliliters (ml) of etanol 97%.The container is then left undisturbed at room temperature for a minimum of three days, with periodic stirring, until all the soluble components have fully dissolved.Subsequently, the mixture is filtered to separate the liquid from the damp solid material called "marc," which is pressed to extract any remaining liquid.The combined liquids are then clarified through either filtration or decantation after being given time to settle.

GC-MS Analysis
GC-MS analysis was carried out so that we could have an idea of the chemicals that were found in the extracts.GC-MS (QP 2010 Shimadzu, Japan: column RTx-5MS (Restek Corp.), length 30 m, inlet temperature, and detector) GC-MS a temperature of 250 degrees, with an operating range of 50-300 degrees.Temperature should be between 50 and 120 degrees Celsius, and the rate of increase should be no more than 4 degrees Celsius each minute.After that, the temperature was raised to between 120 and 300 degrees Celsius, then it was raised once more to a rate of 6 degrees Celsius per minute, and finally, the temperature was held steady for 5 minutes out of a total holding period of 60 minutes.Helium, with a molecular mass range of 50-500, is the standard gas.Reading the area on the GC-MS chart will allow you to obtain an estimated quantitative representation of the molecule.To analyze the GC-MS mass spectrum, the researchers utilized the extensive database of the National Institute of Standards and Technology (NIST).This database contains over 62,000 distinct patterns.The analysis involved comparing the mass spectra of the unknown component with the spectra of known components stored in the NIST library.It was determined what the components of the test materials were called, as well as their molecular weights and structures. .

PCA Analysis
To explore the connections between various variables, a principal component analysis (PCA) was conducted on the entire dataset.The purpose of this analysis was to uncover any underlying relationships or patterns among the variables.(PCA) are two methods that are frequently employed in the process of numerical categorization.[12,13].The principal component analysis (PCA) is a multivariate technique that helps evaluate links between several quantitative variables.This allows for the data to be summarized, linear relationships to be identified, and the number of variables used in regression and clustering to be reduced.Plots of principal components are an extremely helpful tool for conducting exploratory analyses of data [14].

Results and Discussion
The data presented in Figure 1 show the GC-MS test results of Senna alata leaf obtained fromthree different locations, i.e Samarinda, Nyapa Indah, and Samboja.Chemical contents may vary depending on the climate diversity in every research location.Plants will respond in different ways toward various environmental stimuli [15,16].Various secondary metabolites will depend on environmental factors [17].However every coequal species in different locations will still has its distinctive compounds [18].
Senna alata in Nyapa Indah grows in riverside areas, while those in Samarinda and Samboja grow in lowland areas.High intensity light in low altitude growing location will cause alkaloid to be produced intensely (Han et al., 2017).Beside that, the more intense light that the plant receives, the better photosynthesis it will do and the better growth it will get.Low temperature and high humidity can cause the plant to produce low alkaloids (Caradus et al., 2020).Aside from growing location, the other factor that can cause a secondary metabolite's metabolism become different is the soil's type and density/header's closing.The soil where Senna alata grows in Nyapa Indah on the riverside areas tends to be wet all the time with a color that tends to be black, while the soil where Senna alata grows in Samarinda and Samboja tends to be drier.Senna alata on the riverside of the Nyapa Indah tends to get a lot of sunlight because there are no obstructions.In contrast, Senna alata in Samarinda and Samboja tend to compete for sunlight with the surrounding plants.A plant's density/header's closing will affect how intense the light, the temperature, and the humidity will be.A sparse header will cause the light that goes into the field's surface to be more intense, so that it will rise the temperature and lower the humidity.Soil's type/category can also affect the plant's growth quality.Robust soil in general hashigh robustness value that will affect the production of alkaloid contained in a plant [19] High temperature can also be affected by the environment's CO2.High temperature will be a threat to the plant itself, and as a response, the plant will adapt itself to the environment by producing secondary metabolites such as compounds that has antioxidant properties to oppose free radicals in the environment.[20].On higher temperature, as an extra defensive synergy toward the threats in the environment, the plant will produce more flavonoid as well [21].The location's altitude can also affect not only the diversity but also the concentration of the chemical compounds that are contained in the species [22,23].
Wang and colleagues showed that In mice that were given a diet high in fat and fructose, phytol improved their ability to tolerate glucose and increased the number of adipocytes in the inguinal subcutaneous white adipose tissue.These findings were in line with an augmentation of adipogenesis and glucose uptake in the 3T3-L1 cell line.Moreover, it was observed that these effects were linked to the activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt signaling pathway.This pathway is also associated with benefits for weight reduction.[75,76].Other researchers have also conducted studies related to phytol-induced reductions in fatty acid levels to promote lipid metabolism [77,78].

Conclusion
There are variances in the yield of secondary metabolites according on where Senna alata is grown, specifically in the riverside Nyapa Indah region and the plains of Samarinda and Samboja.This research was conducted to estimate the compounds contained in extracts by using GC-MS analysis, and to discover relationships between different variables by using PCA.Based on the results of the GC-MS analysis, Phytol was identified as the primary compound in S. alata from two areas, Samarinda and Samboja.However, in Nyapa Indah, Berau, the main components were found to be 1,2-Benzenedicarboxylic acid and mono(2-ethylhexyl) ester.Nevertheless, S. alata generally contains numerous secondary metabolites, which are valuable as a source of microbicides, pesticides, and various pharmaceutical drugs.Therefore, the leaf extracts of S. alata could potentially serve as a valuable source for traditional medicine, providing a range of pharmaceutical agents.

Figure 2 .Figure 3 .Figure 4 .
Figure 2. GC-MS test results of Senna alata of three locations Figure 3  showed main compound of three locations.Phytol is the main compound in S.alata in two areas, i.e.Samarinda and Samboja.Meanwhile, in Nyapa Indah, 1,2-Benzenedicarboxylic acid, and mono(2-ethylhexyl) ester are the main componentsin the area.