Hepatoprotective And Antidiabetic Effect Of Phyllanthus Extract Biology Essay


Phyllanthus niruri is a well known and widely used herb especially in Asia, which contains several interesting bioactive constituents and possesses health promoting properties. In this report, the hepatoprotective and antidiabetic activities of methanol extracts from the phyllanthus plant are discussed. The hepatoprotective effect of the phenolic content in phyllanthus extract was shown by introducing the phyllanthus extract before and after the hepatic damage was induced by nimesulide, D-galactosamine and alcohol. While the antidiabetic effect of the phyllanthus extract was also studied in animal models with diabetes induced with alloxan and streptozotocin. The results from a few studies reviewed show that phyllanthus extract is able to reverse the effect brought about by the hepatotoxic and diabetogenic agents. The serum marker and antioxidant levels returned to normal while the reactive oxygen species were reduced. The effectiveness of phyllanthus extract is dose dependent. Increasing concentrations of phyllanthus extract (phenolic content) showed an increasing effectiveness of the hepatoprotective and antidiabetic ability on the tested animals or cells. This report also highlights the medicinal potential of the phyllanthus extract and the relationship of the phenolic contents with the hepatoprotective and antidiabetic effect of phyllanthus extract.

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Phyllanthus plant belongs to a plant family known as Phyllanthaceae. Phyllanthaceae is a pantropical family that contains 60 genera and 2000 species. Plant species from this family normally are herbs, shrubs or trees. The sexual expression of the phyllanthaceae plants can be monoecious or dioecious. These plants normally lack latex and contain extra nectaries structure. The leaf arrangement of phyllanthaceae is in such that the leaves are grown singly at different heights on the axis and distichous which regularly arranged leaves one above the other in two opposite rows and one on each side of stem. The leaves are mostly simple where it is only with one blade. The leaf blade is membranceous to coriaceous (tough). It may be toothed, scalloped or lobed but is never divided until the leafstalk. The leaf margins are smooth, curved or straight and they are without teeth or undulations. Rarely, secretory foliar glands are embedded in the surface of plant. The stomata of plant are paracytic or can be anomocytic. The stipules are usually present and persistent (remaining attached). Latex layer is absent in these plant. The stem of the Phyllanthaceae plant can be indumentum (hairy) and less branched. The characteristics of phyllanthaceae's flower are hypogynous, actinomorphic (symmetrical), unisexual, pistillode or staminode can be absent or present. The calyx and corolla are mostly inconspicuous and usually monochlamydeous and rarely dichlamydeous. The characteristic appearance of seeds are trigonous, ovoid, ellipsoid (solid object roughly with an elliptic outline from the side view and circular outline from the end view), lack or rudimentary of caruncle. The coat texture of phyllanthaceae seeds are dry outside but fleshy in the inside where they are thick, firm yet soft plus can be easily sliced while the endosperm can be present or absent (Fiaschi et al., 2005) (Marcos José da Silva. n.d).

Phyllanthus species was originally categorized under the plant family known as Euphorbiaceae but it was then categorized under a new family phyllanthaceae due to some characteristics of the plant. The Euphorbiaceae plant species exhibit similar characteristics as phyllanthaceae. Phyllanthaceae plant species are characterized morphologically by four characteristics which separate them from the Euphorbiaceae species which include the absence of latex, extrafloral nectaries in the leaves, ovary bi-ovulated and seeds without caruncle. Phyllanthaceae species are usually pubescent (hairy but not densely), latex mostly present and caustic, milky and transparent and extra floral nectaries are present. The ovaries are commonly uni-ovulate, fruit are capsular and the seeds are carunculated. This is different from Euphorbiaceae where its species are usually glabrous (hairless), even if hair is present it is usually simple and both the latex layer and the extra floral nectaries are absent. There are normally with two ovules per locule, fruits capsular to drupaceous (containing more than one seeds) and caruncle can be present or absent in the Phyllanthaceae species (Marcos José da Silva. n.d)

"Phyllanthus" species is the largest genus in the plant family of Phyllanthaceae. "Phyllanthus" has a unbelievable diversity of growth forms including annual and perennial herbaceous, arborescent, climbing, floating aquatic, pachycaulous, and phyllocladous. It has wide varieties of floral morphologies, chromosome numbers and has the widest varieties of pollen types of any plant genus. All types of "Phyllanthus" species express a specific type of growth known as "phyllanthoid branching" in which the leaves on the main plant axes on the vertical position are reduced to scales called "cataphylls" while leaves on the other axes (plagiotropic) at the horizontal position, deciduous and floriferous (flower-bearing) develop normally (Fiaschi et al., 2005). Phyllanthus is distributed in all tropical and subtropical regions on Earth for example in South Africa, China, Malaysia, Indonesia, and India. Leaf flower is the common name for all Phyllanthus species. These plants are found growing wild scattered all around the city. Many people treat these plants as useless "weeds" but these "weeds" can grow prolifically by themselves. In fact, these "weeds" turn out to have a wide range of medicinal properties.

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Figure 1: Phyllanthus and its botanical information. (Marcos José da Silva. n.d.)

Figure 1 (Marcos José da Silva. n.d.) shows that basic information of the phyllanthus plant. There are numerous phyllanthus species in the world. The phyllanthus species that grow in Malaysia is Phyllanthus niruri and Phllanthus amarus. Phyllanthus known as Pokok Dukung Anak among the Malaysian because the seeds that grown on the backside of the stem gives an image as the plant is carrying its child. Phyllanthus can be found at road side or near the drain. It needs certain humidity level in order to growth thus this plant unable to found in dried area or country.

Chapter 1.1: Phyllanthus plays an important role in medicinal properties.

As we can see the technology of the world is in a magnificent speed of improvement, it brings many effects to human. The most obvious effect is exposing living organisms to diseases such as cancer, heart diseases, viral diseases and so on which are mostly caused by various environmental factors. A lot of natural resources for examples herbs like Phyllanthus species have been used mainly for medicinal purposes, especially as traditional remedies which have been used by our ancestors hundreds of years ago. Herbs possess unique ways in improving our health.

The Phyllanthus plant is actually rich in polyphenolic compounds. Phenolic compounds are readily found in herbs, vegetables, fruits and other plants and some of the examples of phenolics are flavonoids, carotenoids, nitrogenous compounds and phenolic acids. The phenolic compounds or bioactive agents extracted can cure and prevent certain diseases due to their antioxidant, hepatoprotective and antidiabetic properties. There are several bioactive agents that are isolated from the phyllanthus plant. For example, phyletralin, phyllanthin, methylgallate, rhannocitrin, methyl brevifolincarboxylate and trimethyl-3,4-dehydrochebulate. All these compounds are in yellow powder after extraction. They exhibit excellent effects on health. They can act as antioxidants due to the presence of hydroxyl substituents and their aromatic structure, which give them the ability to scavenge free radicals (VillaËœno et al. 2007). These bioactive compounds isolated from Phyllanthus spp. are present in each and every part of the plant in varying concentrations. The highest concentration can be obtained from the leave (Fang Shih-Hua et al. 2008).

Figure 2: Bioactive compounds isolated from Phyllanthus (Fang Shih-Hua et al., 2008)

When compared to the research done by Markom and coworkers (2007) different bioactive agents were isolated from the phyllanthus plant. Flavonoids such as geraniin, corilagin, elagic acid and gallic acid which are categorized as hydrolysable tannins were isolated from the phyllanthus plants. These bioactive compounds have similar functions in cells but they were isolated with different solvent selections. A few types of extraction, fractionation and purification and screening steps can be chosen in herbal processing.

From the research done by Markom and coworker (2007), the effects of various organic and aqueous solvents with different polarities on the extract yield and the content was investigated. They found out there are few compound that they able to isolate out from phyllanthus plant by using different types of solvent and method of extraction (solvent extraction and high pressure extraction) such as hydrolysable tannins, namely gallic acid, ellagic acid and corilagin as shown in Figure 3.

Figure 3: Chemical Structures of Hydrolysable Tannins (Markom et al.2007)

Chapter 1.2: Safety concerns of consuming Phyllanthus as medicine

Herbal medicines are very popular in developing and underdeveloped countries. Phyllanthus is used as a folk medicine for jaundice and other diseases in Malaysia and other countries. Even though a large number of clinical trials have been done on the benefits of P. amarus, so far no systematic toxicological investigation has been reported on this plant, especially with P. amarus growing in Malaysia. Since the efficacy of Phyllanthus species varies with geographical locations and varieties and because of the variations in the composition of various constituents, the bio-safety of using P. amarus grown in Malaysia as medicine for human consumption has to be ascertained by conducting acute and chronic toxicity studies. Understanding the potential adverse effect of herbs used by human is necessary for implementing safety measures in the public. In the case of P. amarus, available reports are generally unanimous on the efficacy of the plant grown in different countries but no systematic safety study had been done so far and hence Sirajudeen et al. (2006) carried out a toxicity study of the plants. Aqueous extract of leaves of locally grown P. amarus was administrated orally to rats, then morphological, biochemical and histological changes of rat liver were assessed. The toxicity of the phyllanthus extract toward living cells was determined by measuring the level of total protein and activities of serum marker enzymes of control and P. amarus administered groups of rats. From the results obtained, group I which was the control group (without administration of phyllanthus extract) showed similar levels of total protein and activities of serum marker enzyme compared to Groups II, III and IV which were the phyllanthus extract administered groups at the doses of 100 mg/kg body weight/day, 400 mg/kg body weight/day and 800 mg/kg body weight/day for six weeks, respectively (Sirajudeen et al., 2006). This shows that by consuming the plant extract alone does not give any significant biological changes to the protein and serum marker enzyme level.

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Any elevation of protein and serum marker enzyme levels (ALT, AST, ALP and LDH) may indicate the occurrence of liver damage. From the results obtained from study done by Sirajudeen and coworkers (2006), it is shown that ingestion of phyllanthus extract did not alter the levels of protein and serum marker enzymes (ALT, AST, ALP and LDH). In the chronic toxicity study, the non-toxic nature of P. amarus extract administration was confirmed by biochemical analysis and histological studies for example, by using light microscopy, by proliferative cell nuclear antigen study and apoptotic study on rat livers. From the chronic study, no significant difference was observed between the control and P. amarus extract administered to male and female rats in the total body weight gain as well as in the liver marker enzymes analyzed in serum. The results did not show significant changes between control and P. amarus extract administered rats. Therefore, acute oral administration of P. amarus extract is non-toxic to the rat liver because even at a dose of 5 g /kg body weight and also the chronic toxicity studies of P. amarus extracts administration showed the absence of cumulative toxicity as reflected by the non-significant change in the parameters studied as well as from the results of the histological studies. This clearly indicates the non-toxic nature of the plant extract. Toxicologists agree that any test substance that is not lethal on acute administration at a concentration of 5g/kg body weight, is essentially non-toxic (Sirajudeen et al., 2006).

This can be further proven by the research done by Suresh and coworkers (2008) on the acute toxicity evaluation of extract in Wistar rats (Suresh et al., 2008). To determine acute toxicity, a group of six animals were fed with a single dose of the extract at a dose of 2000 mg/kg which was ten times that of therapeutic dose. Another group of six animals was kept as normal control. Observations were made systematically at 1, 2, 4, 8, 12, 24, 48 hours after administration of phyllanthus extract and changes related to skin, fur, eyes, body temperature, body weight, and behavior and enzyme excretions. Animals were observed for a period of 14 days to see whether if there was any mortality. After 14 days animals were sacrificed, serum was separated and analyzed for ALT, AST, ALP and LDH cholesterol, triglyceride, creatinine, urea and hemoglobin were also analyzed. The rats were dissected and changes in internal organs were monitored. No mortality was observed and animals did not show any significant changes in skin, fur, eyes, body temperature, body weight, internal organs, behavior and in the nature of excretions in the toxicity study. The serum parameters of toxicity analysis showed that there were no significant changes between control and extract treated groups. It is suggested that the extract did not bring any significant toxicity even at a high extract dose. (Suresh et al., 2008)

Chapter 1.3: Is intraperitoneal Administration better than Oral Administration of Phyllanthus Extract?

The route of administration of medicine can be varied with types of medicine. The route or course for the active substance or chemical taken from the uptake or application location to the targeted location where it begins to shows the hepatoprotective and antidiabetic effect is the focus of some studies. Oral administration is a convenient, although not always equally effective way of drug administration route. Kinetics of drug uptake and distribution might be very different from those drugs that are given by intraperitoneal administration (Chatterjee et al., 2007). The effect of methods and the difference in effectiveness of administration of phyllanthus spp. on hepatoprotective function were looked into by Chatterjee and coworkers (2007). They monitored the level of serum marker enzymes level after administration of phyllanthus extract through oral and intraperitoneal injection. The results were tabulated and both administrations of phyllanthus extract to the rats were able to reduce the hepatic damage done by the nimesulide. The levels of serum marker enzyme (GPT, GOT and ALP) in mice liver through oral administration was higher compared to result obtained by intraperitoneal administration (Chatterjee et al., 2007). The levels of antioxidants (SOD, CAT, GSH, and MDA) increased when phyllanthus extract was given through intraperitoneal administration. This shows that phyllanthus extract being administrated to the mice through intraperitoneal injection gave a higher hepatic protective effect. The reason of this to occur is due to the presence of some protein molecules in the herb that might be involved in its hepatoprotective action. Research of Chatterjee and coworkers (2007) suggested that some protein molecules in the extract might be degraded in the stomach, but intraperitoneal administration keeps them intact and helps them to exhibit their function properly. On the other hand, it is possible that heat treatment or enzymatic digestion destroyed the biological activity of the extract (Chatterjee et al., 2007).

Chapter 1.4: Different types of extraction give different effectiveness of phyllanthus in medical properties

Some herbs or crops are perishable in their fresh state and may start to deteriorate within a few days after harvest. In order to preserve these plant products the best way is to dry them so that the qualities of important products can be conservee, storage volume reduced and the shelf life extended. Drying will inactivate the enzymes polyphenol oxidases (compounds that protect plant from internal damages) and can either be performed by traditional sun drying or a modern way of microwave drying and oven drying. However, drying fresh plant tissues may lead to significant changes in the composition of phytochemicals (chemicals that may affect health but are not essential nutrients) because the enzymatic pathway or process was disturbed by the heat. There are numerous reports that stated the methanol extracts of powdered air-dried phyllanthus plant showed high antioxidant activities (Lim and Murtijaya. 2007). However, there was not much information regarding its antioxidant properties as affected by various drying methods and aqueous extracts. Therefore, this chapter is to evaluate the effect of various drying methods on the total phenol contents and antioxidant properties of alcoholic and aqueous extracts of the phyllanthus plant.

In the research done by Lim and Murtijaya (2007), they utilized three different drying methods which included sun-dried, microwave dried and oven dried extracts resulted in weight loss of 72.2% due to loss of water. Drying of the plant materials caused them to become crisp in nature, thus making them easier to grind during extraction. All dried plant parts turned light brown, except those dried with microwave which turned slightly darker green in color. The antioxidant, hepatoprotective and antidiabetic effect sof phyllanthus extracted by various methods were measured by determining the total phenolic content (TPC), DPPH radical scavenging activity assay and Ferric reducing antioxidant power (FRAP) assay.

Table 1: Effects of processing on the TPC and antioxidant activity of P. amarus extracted with various solvents

*value in bracket indicates that the plant sample was processed

(Lim and Mutrajaya. 2007)

Table1 shows that TPC and DPPH radical scavenging activity of fresh phyllanthus plant materials were dried and extracted with different solvents (100% methanol, boiling water and cool water). From the results, different drying treatments and various solvent extracts affected the TPC and subsequent antioxidant activities of phyllanthus extracts. Decrease in TPC and antioxidant activity were exhibited by the reduction in both DPPH free radical scavenging activity (higher IC50 and lower ascorbic acid equivalent antioxidant capacity AEAC) and ferric reducing property (lower FRAP values).Drying caused significant decrease of total phenolic content in phyllanthus extracted using methanol especially extract using microwave drying which caused the highest TPC loss of 59% compared to fresh samples. On the other hand, the TPC of methanol extracts of oven-dried and sun-dried samples only dropped by 23% and 19%, respectively. There are explanations why both the TPC and antioxidant activity of the extracts decreased. There was a loss of TPC when sun drying method was used for extraction; the reason for this to occur is due to enzymatic processes that occurred during sun drying. The heat energy that obtained from sun drying method did not immediately deactivate degradative enzymes (polyphenol oxidases) in the plant material. Therefore, before they are denatured by the heat they were able to degrade phenolic compounds before the plant materials were dried. Oven heating at 50 0C resulted in the second lower TPC value, the processing rapidly inactivated polyphenol oxidases present in plant materials; however, initial activities of the enzyme may have occurred earlier and caused some polyphenols to be degraded. Microwave heating resulted in the highest TPC loss, in this method the microwave energy was absorbed by water molecule, this supplied more energy than the traditional way of heating and was able to inactivate degradative enzymes very much faster than oven heating and a drastic loss of TPC was observed. This indicates that heating treatments not only deactivate enzymes but also are able to degrade phytochemicals in the plant material. The heat generated from microwave is intense compared to solar radiation which resulted in higher loss of TPC. According to Lim and Murtajaya (2007), some phenolic compounds decomposed rapidly when exposed to direct sunlight or dried at increasing temperature. Drying process would result in a depletion of naturally occurring antioxidants in plant materials (Tomaino et al., 2005). Intense and prolonged heat treatment will lead to denaturation of natural antioxidants because these compounds were relatively unstable. However methods of processing may not always affect the compositions. For example, heat causes little or no change to the content and activity of naturally occurring antioxidants, such as carotenoids which is not degraded even after intense or prolonged heat treatment due to its heat stable characteristic. As a conclusion, data obtained indicate that drying process of the plant materials will cause the reduction of TPC values, depending on which method is used.

In the study of Lim and Murtajaya (2007), different types of solvent were used for extraction of the plant material. Three solvents were used which included boiling water, cool water and methanol. Methanolic extracts of phyllanthus plant possessed both higher TPC and antioxidant activity than cool water extracts because methanol was able to denature polyphenol oxidases. Methanol as an organic and volatile solvent can efficiently degrade the cell walls of the plant and therefore was able to extract more phyllanthus endocellular materials (phenolic compounds) than water. However, results showed that only processed plant materials extracted by methanol led to a decrease in both TPC and antioxidant activity. While for boiling water extraction of phyllanthus plant, results showed higher TPC in phyllanthus extract than plant material extracted using methanol. Boiling water also extracted significantly higher TPC from both fresh and processed plant materials than cool water. The higher TPC extracted in boiling water gave rise to strong radical scavenging activity in DPPH assay as exhibited by the lower IC50 and higher ascorbic acid equivalent antioxidant capacity (AEAC values). Boiling water and methanol were able to inactivate polyphenol oxidase which is the degrative enzyme present in fresh plant materials that caused the higher yielding of TPC than cool water. However, plant extracted with methanol yielded a lower TPC than both boiling and cool water. This may be due to the presence of certain very polar compounds in dried plant materials, which can only be extracted with very polar solvent (water). When comparing the extraction efficiency of boiling and cool water, extraction using cool water yielded significantly less phenolic content than boiling water from both fresh and dried P. amarus plant materials. Extraction by boiling water resulted in higher yield of polyphenol compounds from dried plant materials than cool water, this indicated that the heat from boiling water was one of the factors that led to higher total phenols extracted from dried plant samples. This is supported by the research done by Toor and Savage (2006) where they showed that intense heat from boiling water was able to release cell wall phenolic compound or bound phenolic compound due to the breakdown of cellular component, thus causing more polyphenolic compounds to be extracted (Toor and Savage, 2006). Research done by Markom and coworkers (2007) showed that phyllanthus was most soluble in polar solvents such as water and aqueous ethanol. Low extract yields were obtained in the non-polar solvent such as n-hexane (1.8%) and petroleum ether (2.2%). This shows that solvent polarity plays an important role in plant component extraction process (Markom et al., 2007).

Thus far, the most frequent method used to extract bioactive agents from phyllanthus is methanolic extract. This is because alcohol can make the plant material porous and allow the content in the plant to be extracted faster compared to normal water as solvent. The most important thing is that most components in the phyllanthus plant are hydrophilic or water-soluble. Methanol can give yield at higher rate which provides advantages in larger quantity at a shorter time because it is a polar organic solvent that is able to denature the degrative enzyme and degrade the cell wall of plant sample. Thus, this explained why methanolic extraction is used more frequently compared to extraction with normal solvent like water.

As a conclusion, data on the effects of drying on TPC and antioxidant activity of herbs are conflicting due to several factors. These include the different drying conditions and types of extraction solvents. The screening by various extraction solvents showed that most components in phyllanthus plant are hydrophilic or water soluble. Since most medicinal herbs are prepared for consumption using aqueous methods, determination of herbal antioxidant levels should also be based on extraction using boiling water rather than just on methanolic or ethanolic solvent alone (Lim and Mutrajaya. 2007)

Chapter 2

Hepatoprotective effect on phyllanthus on Liver

In this modern century, issues related to health have been given an important attention. Based on the information obtained from World Health Organization, liver related diseases have been categorized as one of the world's leading killer diseases. Liver disorders can be caused by tonnes of different agents like alcohol consumption, environmental toxins and viruses. All these had become threats to public health. Currently the importance of herbal medicine in curing various disorders or diseases had been established. There are lots of potential herbal medicines available for liver disorders in various parts in the world (Suresh et al., 2008).

Chapter 2.1: Introduction to hepatotoxins

First of all, the knowledge to our liver is indispensable. Our liver is actually the largest internal organ and plays a central role in the entire body. Liver has an important role in regulating nutrient levels in the body. The most important function of liver is that it is the primary site for activation, clearance, detoxification and excretion of drugs or toxins that enter our body. There were numerous elements that can cause liver damage, for example, environmental toxins, hepatotoxins, alcohol consumption and viruses. In this review, liver damage caused by hepatotoxins is highlighted. Hepatotoxin is any chemical that can cause damage to or disease in the liver. Figure 2 shows the examples of the hepatotoxins that can lead to hepatic damage.

Figure 4: Examples of hepatotoxins

Nimesulide is one of the non-steroidal anti-inflammatory drugs ( NSAIDs ) that possess analgesic (pain killer or relieve pain), antipyretic (reduce fever) and anti-inflammatory properties. Thus it is used as treatment of acute pain. NSAID is a Cyclooxygenase 2 (COX2) selective inhibitor that inhibits cyclooxygenases which leads to a decrease in prostaglandin production and leads to reduction of pain and also inflammation. On the other hand, it also inhibits the leucocyte function. However, this drug had been removed from the market due to its hepatotoxicity to the consumers. Nimesulide can cause several side effects to the consumers or users if they consumed it for a long term. Nimesulide can cause liver injury thus it is not suitable to be used as primary therapy as analgesic and antipyretic. A study revealed that five-day use of nimesulide (by a 70 year old woman) will cause jaundice. The effect can also be seen through an action on COX-2 colon cancer that occurs. There are some minor effects that may experience by consumers where they may encounter headache, skin rash, vomiting and dizziness (Stadlmann et al., 2002).

D-Galactosamine ( D-GaIN ) is a hepatotoxin agent that causes liver injury similar to acute hepatitis. D-Galactosamine may cause increasing radicals in the body such as melondialdehyde which is mutagenic. It is thought to induce hepatotoxicity by inhibiting the synthesis of RNA and protein through a decrease in cellular UTP concentration, which finally leads to the necrosis of liver cells (Seckin et al., 2008). D-GalN will also induce apoptosis (programmed cell death) in liver. Whenever DNA fragmentation is detected in the blood serum, it indicates the presence of apoptotic cell death. When D-galactosamine induced apoptosis the concentrations of tumor necrosis factor will increase which is the agent that cause apoptosis of liver cell (Seckin et al., 2008).

Alcohol is categorized as a psychoactive drug that has depressant effect. Alcohol produces toxic chemicals like acetaldehyde which can damage liver cells. In the liver, alcohol dehydrogenase oxidizes ethanol and converts it into acetaldehyde, which is then further oxidized into harmless acetic acid by acetaldehyde dehydrogenase which involves reduction of NAD+ to NADH. Long term consumption of alcohol can lead to alcohol diseases because of accumulation of aldehyde


Phyllanthus extract is a traditional herb that widely had been widely used in Asia. It had become an important economic healthy food. From this literature review we can see that there is research that shown magnificence effect on hepatoprotective and antidiabetic effect. There are also researches showed that phyllanthus extract is safe to be consumed. The effect of phyllanthus extract on hepatoprotective and antidiabetic effect is dose dependant. Why not each and everyone starts to plant this small little phyllanthus plant around us and show other people that this small plant can bring healthy to all of us.