Study on Centella Asiatica Extract Dosage Form

The Chinese herb centella asiatica was first recorded in the Divine Husbandman's Classic of the Materia Medica and has been used externally and internally for more than 2,000 years. Centella asiatica is the dried whole herb of the plant Centella asiatica (L.) Urb. in the family Apiaceae. It grows in shady, damp meadows or along the edges of ditches. It contains the pentacyclic triterpenoids asiaticoside, madecassoside, asiatic acid and madecassic acid, as well as flavonoids such as quercetin and kaempferol. Centella asiatica[1] is bitter, pungent and cold in nature, entering the liver, spleen and kidney channels. It has the effects of clearing away heat and dampness, detoxifying and reducing swelling. It is often used to treat damp-heat jaundice, heatstroke diarrhea, stone dysuria, carbuncle and swelling caused by poison, and injuries from falls and blows. Domestic and foreign researchers have further specified the pharmacological activity of Centella asiatica through animal experiments or clinical trials, and have sought to achieve better clinical results by preparing new preparations of Centella asiatica-related ingredients.

1 Pharmacological activity

The chemical components of Centella asiatica mainly include asiaticoside, madecassoside, asiatic acid, and madecassic acid. Asiatic acid and madecassic acid are the aglycones of asiaticoside and madecassoside, respectively. See Figure 1.

1.1 Antioxidant stress effect

Abdul Hisam EE et al. [2] studied the effect of a combined extract of moringa and centella asiatica (TGT-PRIMAAGE) on hydrogen peroxide (H2O2)-induced oxidative stress in human dermal fibroblasts. They found that TGT-PRIMAAGE can effectively reduce H2O2-induced production of reactive oxygen species. In cells treated with TGT-PRIMAAGE, the activities of superoxide dismutase and catalase increased, and the content of malondialdehyde decreased significantly. This indicates that TGT-PRIMAAGE can protect cells from H2O2-induced oxidative stress and prevent H2O2-induced cell aging.

Lin Chenxi et al. [3] found that asiaticoside can reduce fatty lesions and alleviate liver damage in a hyperlipidemia model of golden hamsters. The mechanism may be related to enhancing the antioxidant effect of the liver and reducing blood lipids and liver lipids.

Mai Langjun et al. [4] found that asiaticoside can reduce inflammation and injury in the lungs of newborn rats caused by hyperoxia, and improve the symptoms of bronchopulmonary dysplasia, and there is a dose-dependent effect. It is speculated that the mechanism of action may be related to downregulating the expression of miR-155 and upregulating the expression of signal inhibitor of cytokine-1 (SOCS-1) in peripheral blood cells.

1.2 Promoting tissue healing and improving microcirculation

Chiaretti M et al. [5] studied the therapeutic effect on chronic anal fissures and found that compared with traditional treatments, patients treated with Centella asiatica experienced early healing and pain relief, with the flavonoids having the best therapeutic effect. Sastravaha G et al. [6-7] studied the effect of Centella asiatica and pomegranate peel combined extracts on periodontal healing in adult periodontitis patients after scaling and root planing, found that topical administration of Centella asiatica and pomegranate peel extracts plus scaling and root planing can significantly improve the clinical symptoms of patients with chronic periodontitis. Paocharoen V [8] found that Centella asiatica extract has the effect of promoting wound healing, can inhibit diabetic wound scarring, and shorten the healing time of diabetic wounds.

Hu S et al. [9] found that oral Centella asiatica preparations can improve local skin microcirculation, increase skin thickness, promote collagen production, improve elasticity, and repair skin damage during pregnancy. SHEN X et al. [10] found that asiaticoside can inhibit pro-inflammatory cytokines, promote skin hydration, promote hyaluronic acid secretion, and maintain skin homeostasis. and has potential medical and cosmetic applications. Cesarone MR et al. [11-13] found that the total triterpenoid fraction of Centella asiatica (TTFCA) has therapeutic and ameliorative effects on venous hypertension microvascular lesions and microcirculation, and has a positive effect on stabilizing low-echo, low-density carotid artery plaques. Incandela L et al. [14] found that the total triterpenoid fraction of Centella asiatica has a therapeutic effect on diabetic microangiopathy, neuropathy and edema.

1.3 Enhances cognitive function and neuroprotection

Wong JH et al. [15] found that Centella asiatica (CA) extract can enhance cognition and increase synaptic occurrence, improving learning and memory. Pharmacokinetic studies of CA have also shown that the bioactive components of CA have low lipid solubility and poor ability to cross the intestinal membrane, further reducing its oral bioavailability. However, it is worth noting that madecassoside and asiaticoside can reach various target organs, especially the brain, within 1 h after a single administration, and remain in brain tissue until the 4th hour of observation, suggesting that the active components of Centella asiatica have a cumulative effect in the body.

Alzheimer's disease (AD) is a neurodegenerative disease associated with cholinergic dysfunction and impaired redox homeostasis. D-gal is an aging agent, and aluminum is a neurotoxin known to be associated with the pathogenesis of AD. Chiroma SM et al. [16–18] used a combination of D-gal and aluminum chloride (AlCl3) to prepare an animal model of AD and to explore the protective effect of Centella asiatica on the cognition and brain ultrastructure of rats induced by D-gal and AlCl3. The results showed that D-gal and AlCl3 significantly impaired the behavior and cognitive function of rats, caused damage to pyramidal neurons in the CA1 region of the hippocampus, and caused morphological changes in the ultrastructure of the hippocampus. Centella asiatica alleviated cognitive dysfunction in rats by restoring cholinergic function, reducing oxidative stress, and preventing morphological abnormalities.

Ar Rochmah M et al. studied the effects of Centella asiatica ethanol extract (CA) on the levels of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10) and the levels of silent information regulator 1 (SIRT1) and brain-derived neurotrophic factor (BDNF) in the hippocampus of rats with chronic stress [19]. TNF-α, IL-10, SIRT1 and BDNF were measured in the experiment using enzyme-linked immunosorbent assay. It was found that the TNF-α level in the stress control group was significantly higher than that in the non-stress control group. Under all stress conditions, the rats receiving the highest dose of CA had the lowest average TNF-α and the highest average BDNF. There was no significant difference in IL-10 and SIRT1 levels between the stress control group and the non-stress control group. It is speculated that the lower TNF-α and higher BDNF may promote the neuroprotective effect of Centella asiatica on rats with chronic stress. Yadav MK et al. [20] found that Centella asiatica extract has the neuroprotective effect of inhibiting acetylcholinesterase activity and promoting spatial memory formation. Wattanathorn J et al. [21] found that Centella asiatica has the effect of alleviating cognitive function and mood disorders in healthy older adults.

1.4 Antitumor effect

Soyingbe OS et al. [22] used agar hole diffusion and microplate dilution methods to determine the antibacterial activity of Centella asiatica extract. The results showed that the extract had good antibacterial and antiproliferative effects on the selected cancer cell lines in the study, suggesting that Centella asiatica extract can be used as an immune enhancer to prevent infections in immunosuppressed cancer patients.

Elevated MAO-B levels can lead to neurodegenerative disease. Subaraja M et al. [23] evaluated the effect of asiaticoside (AD) on the levels and activity of monoamine oxidase A and B (MAO-A and MAO-B). They found that AD had a negative effect on the content and activity of MAO-A and MAO-B. This characteristic is of great significance for the prevention and treatment of neurodegenerative diseases such as Parkinson's disease.

1.5 Anti-anxiety effect

Jana U et al. [24] found that Centella asiatica can significantly reduce anxiety-related diseases, and can also significantly reduce stress and related depression. In addition, it was found that Centella asiatica further improves students' adaptability and willingness to learn, thus indicating that Centella asiatica can be used as an effective drug for the treatment of anxiety disorders and is expected to become a promising anti-anxiety drug in the near future.

1.6 Anti-inflammatory effect

Viswanathan G et al. [25] investigated the protective effect of Centella asiatica methanol extract (CAM) on mouse brain and astrocytes by administering CAM and paracetamol in combination. The results showed that CAM reversed the production of free radicals and reactive nitrogen induced by paracetamol, and increased clearance activity, which was more pronounced at high doses. CAM inhibited the damage caused by acetaminophen to primary mouse astrocytes by inhibiting the expression of pro-inflammatory cytokines and significantly increasing the expression of anti-inflammatory cytokines.

Liang et al. [26] found that primary cultured astrocytes stimulated by lipopolysaccharide (LPS) significantly increased the release of NO and the expression of inflammatory factors TNF-α, interleukin-1β (IL-1β) and interleukin-6 (IL-6) compared to the blank control group. After intervention with asiaticoside, the release of NO and the expression of TNF-α, IL-1β and IL-6 were down-regulated in a concentration-dependent manner. It was concluded that asiaticoside can effectively inhibit the release of NO induced by lipopolysaccharide in astrocytes and down-regulate the expression of related inflammatory factors, suggesting that asiaticoside has a good effect in improving cell inflammatory damage.

1.7 Anti-diabetic effect

Oyenihi AB et al. [27] investigated the effects of Centella asiatica extract on key enzymes in glucose and glycogen metabolism in skeletal muscle in type 2 diabetic rats. After oral administration of a vehicle, Centella asiatica (500, 1000 mg/kg) or metformin (300 mg/kg) for 14 consecutive days in diabetic rats, The activities of hexokinase (HK), phosphofructokinase (PFK) and fructose 1,6-bisphosphatase (FBPase) in skeletal muscle were measured using spectrophotometry, the activities of glycogen synthase (GS) and glycogen phosphorylase (GP) were measured using radiochemistry, and skeletal muscle histology was examined. It was found that compared with non-diabetic rats, the activities of HK (25%), PFK (88%) and GS (38%) were reduced in type 2 diabetic rats. After oral administration of Centella asiatica (500 mg/kg), the activities of PFK (7 times) and FBPase (23%) were increased in type 2 diabetic rats, GS (27%) and GP (50%) activities increased after oral administration of Centella asiatica (1000 mg/kg), and there was no significant change in GS and GP activity after oral administration of Centella asiatica (500 mg/kg). The results suggest that Centella asiatica may lower blood glucose levels and increase skeletal muscle glycogen content in rats. In addition, the study found that oral administration of Centella asiatica in type 2 diabetic rats inhibited the morphological damage to skeletal muscle fibers.

Forte R et al. [28-29] studied the therapeutic effect of flavonoids in combination with Centella asiatica and luteolin on diabetic macular edema without macular thickening, and found that the combination of the three orally administered was beneficial for diabetic macular edema patients to retain retinal sensitivity.

1.8 Anti-ischemic reperfusion injury effect

Wang Di [30] found that asiaticoside has a protective effect on renal ischemia-reperfusion injury in rats. It is speculated that asiaticoside may do so by upregulating the expression of serum superoxide dismutase (SOD) in renal tissue, downregulating the expression of malondialdehyde (MDA), and by upregulating the expression of B-lymphocyte tumor-2 gene (Bcl-2), inhibiting the expression of BCL2-Associated X (Bax) and cysteine protease (Caspase-3), it protects against renal ischemia-reperfusion injury.

2 New preparations

2.1 New preparations of asiaticoside

Centella asiatica total glucosides (CTG) is an extract of Centella asiatica. The active ingredients are triterpene saponins and their derivatives, of which asiaticoside and madecassoside have the highest content and the strongest activity. However, because CTG is mostly composed of polar macromolecules, it is not easily absorbed through the skin epidermis or the intestinal tract to reach the lesion site, so its efficacy and application are somewhat limited. The new formulation provides a solution to this problem.

2.1.1 Liposomes

Liposomes[31] are spherical bodies formed by one or more concentric or non-concentric lipid molecular membranes dispersed in water. The structure of liposomes is similar to that of cell membranes. When used to encapsulate asiaticoside, they can be used as a drug carrier to deliver the drug to a specific site and slowly release the drug. They have a significant permeation-promoting effect and a long-acting sustained-release effect, which is beneficial to improving the bioavailability of the drug. They are an ideal topical skin drug delivery vehicle.

Dong Jiao Jiao [31] found that unmodified madecassoside liposomes have a liquid-like fluidity. When applied to burns or scalds, they have poor adhesion and are prone to drug loss, increasing the frequency of dressing changes. To solve this problem, Dong Jiao Jiao and others synthesized a temperature-sensitive material PEG-PCL-PEG (PECE), In the preparation of madecassoside liposomes by the secondary emulsification method, EPC and PECE with different mass ratios were dissolved in the organic phase together as membrane materials. Finally, PECE-modified madecassoside liposomes with good skin adhesion, drug utilization and significant promotion of wound healing were selected.

Chen Jingyi et al. [32] prepared three preparations of asiaticoside liposomes, D-mannose-asiaticoside liposomes, and stearylamine-asiaticoside liposomes. They found that after asiaticoside was modified into a liposomal preparation, the sustained release of asiaticoside was significantly enhanced, and the targeting effect was enhanced. Chen Jingyi et al. [33] also used natural polyelectrolytes as the capsule material and asiaticoside as the model drug to prepare a new type of liposome-encapsulated capsules using cationic liposomes as templates. The asiaticoside liposome-encapsulated capsules obtained had a high encapsulation rate and good reproducibility.

The study found that in order to ensure the application of liposome-encapsulated capsules in the biomedical field, their preparation needs to be carried out under mild process conditions to ensure the biological activity of cells or proteins as much as possible. Liposome-based liposome encapsulation eliminates the nucleation step, reduces the toxicity or interactions caused by nucleation reagents, and avoids damage to the encapsulation structure by external forces. At the same time, the particle size of the liposome-based liposome encapsulation prepared using liposomes is relatively small, the drug loading is relatively high, and the finished liposome has a gel-like viscoelasticity. Chitosan is biocompatible, has low toxicity and is biodegradable, so it is selected as the material for the polyelectrolyte microcapsules.

Yan Dan et al. [34] optimized the preparation process of Centella asiatica total glucoside (CTG) liposomes and found that when the mass ratio of lecithin to cholesterol was 4:1, the mass ratio of lecithin to CTG was 23.22:1, and the volume ratio of the organic phase to the aqueous phase was 7:1, the resulting liposomes were spherical in shape, with a smooth surface, no adhesion, and good stability. The CTG liposome has a high encapsulation rate, small particle size, high skin retention, and obvious sustained-release effect.

2.1.2 Microspheres

Porous microspheres[35] are a type of microspheres with a porous structure, also known as microsponges. The interior of the microspheres is filled with intercrosslinked pores, which have a high drug loading capacity, can improve stability, reduce side effects, and regulate the slow release of drugs at the smallest effective dose. They have excellent skin affinity, are not prone to the growth of bacteria and microorganisms, and are safer to use because they are larger in size and cannot penetrate the epidermis.

Xie Shengyang [35] used the solvent diffusion method to prepare optimized porous microspheres with a good drug encapsulation rate and excellent particle morphology. Compared with the drug solution, the cellular uptake rate of the drug by the porous microspheres was increased by 5.6-9.1 times. In vivo wound healing experiments have shown that Centella asiatica polysaccharide porous microspheres have a significant effect in promoting wound healing. A study of the histopathology of rat skin showed that Centella asiatica polysaccharide porous microspheres can significantly promote the regeneration of collagen and skin appendages. The results also show that ethyl cellulose microspheres have an efficient loading and sustained-release effect on asiaticoside, which can improve the stability of asiaticoside, increase its cellular uptake, and improve its effect of accelerating skin damage repair and regeneration.

2.1.3 Microemulsion

Microemulsions[36] can change the structure of cell membranes and improve the permeability of the stratum corneum due to the presence of an oil phase, emulsifier and co-emulsifier, and have the effect of promoting transdermal absorption of drugs.

Xiong Huimin et al. [36] optimized the prescription of Centella asiatica saponin microemulsion. The obtained Centella asiatica saponin microemulsion was round and regular in shape, with a relatively uniform particle size distribution. A comparison showed that the cumulative transdermal drug permeation of Centella asiatica saponin microemulsion and microemulsion gel within 12 h was significantly higher than that of Centella asiatica saponin cream ointment. The amount of drug retained in the skin was 3.3 and 6.7 times that of asiaticoside cream ointment, respectively. It was concluded that asiaticoside microemulsion gel can significantly increase the cumulative transdermal drug delivery and improve the retention of the drug in the skin, and is expected to become a new topical drug delivery system for asiaticoside.

2.1.4 Nanomilks

Nanomilks[37] are used increasingly in transdermal drug delivery research as drug carriers, as they have the advantages of increasing drug solubility, improving skin affinity, reducing the skin barrier effect, increasing the transdermal rate of the drug, and increasing the amount of drug retained in the skin.

Peng Qian et al. [37] prepared asiaticoside nanomilks (ASI-NEs) and nanobullets (ASI-NBGs), and compared their in vitro transdermal properties with those of a commercially available ointment. The in vitro transdermal results showed that after 12 h of transdermal administration, the cumulative per unit area permeation of ASI-NEs and ASI-NBGs was 6.57 and 2.23 times that of the commercially available ointment control group, the skin retention was 5.93 and 4.48 times that of the commercial ointment control group, respectively. HE staining showed that after using ASI-NEs and ASI-NBGs, the epidermal structure was basically intact, the stratum corneum was loose and thin, the keratin fragments increased, the spaces between the cells of the spinous layer increased, and the cells of the basal layer were loosely arranged.

Laser scanning confocal microscopy (CLSM) studies showed that the ASI-NEs labeled with the fluorescein isothiocyanate (FITC) fluorescent probe penetrated the skin quickly, and were evenly distributed in the dermis 6 h after application. The fluorescent area and integral optical density (IOD) were 28.81 and 32.51 times those of the FITC aqueous solution control group, respectively. The results show that the prepared ASI-NEs and ASI-NBGs have good transdermal properties. The transdermal mechanism is mainly to destroy the microstructure of the stratum corneum and use the appendages of the skin to transport the drug through the skin to exert a therapeutic effect. The study also found that NBGs can improve the fluidity of the nanoemulsion, prolong the residence time of the nanoemulsion on the skin, increase the stability of the drug, and improve the transdermal absorption rate. It is a new transdermal drug delivery system with broad application prospects.

2.1.5 Gels

Gel ointments [38] are made by coating a hydrophilic gel matrix onto a non-woven fabric or other backing material. Not only is the paste uniform and non-greasy, but the number of administrations is also reduced, which greatly improves patient compliance. Cui Xiaojun [38] used NP700 as a base prepared Centella asiatica gel ointment with Centella asiatica total glycosides and aloe vera gel as the base, and compared with commercially available drugs, it was found that the release rate of the Centella asiatica gel ointment was faster, the in vitro release kinetic process was more in line with the Higuchi model, and it had a better scar healing effect. At the same time, the addition of aloe vera also effectively accelerated the time of scar healing.

2.2 Centella asiatica acid derivatives

Centella asiatica acid is a multifunctional active ingredient, but its application is limited due to its low solubility, low bioavailability, and difficulty in crossing the blood-brain barrier. Some researchers have modified the structure and improved the properties of centella asiatica acid to obtain derivatives with higher bioavailability and better activity, which is of great significance for the application and development of centella asiatica acid [39].

Li Xiaoxiao [40-41] used asiatic acid as a lead compound and adopted a computer-aided drug design method. By simulating the docking of the Survivin protein and small molecule inhibitors, the active group was extracted and introduced into the A ring of asiatic acid. At the same time, the C-28 position structure was modified to obtain a series of asiatic acid derivatives. After eliminating compounds with high toxicity and difficulty in synthesis, 12 centella acid derivatives with novel structures and outstanding activity were virtually screened, and it was found that the anti-tumor activity of the synthesized compounds was superior to that of the parent centella acid.

Zhu Bing et al. [42] synthesized the 3,23-O-isopropylidene asiatic acid (A1) derivative by introducing an isopropylidene group between the 3rd and 23rd positions of asiatic acid, and the asiatic acid benzyl ester (Z3) derivative by introducing a benzyl group at the 28th position. After these two derivatives were administered to rats by gavage and compared with the pharmacokinetic parameters after gavage with asiatic acid, it was found that the absolute bioavailability of A1 was 4.67 times that of asiatic acid, and the pharmacokinetic properties were superior to those of asiatic acid, making it worthy of further development and research.

Feng Zhonghua et al. [43] synthesized six new madecassic acid nitrogen derivatives by modifying the structure of madecassic acid. Madecassic acid itself has a certain antitumor activity, and the madecassic acid nitrogen derivatives obtained after modification are expected to exhibit stronger antitumor activity.

3 Discussion and conclusion

In the current research on new Centella asiatica preparations, the pharmacological activity of Centella asiatica glycoside preparations has mostly focused on transdermal absorption, while there have been relatively more studies on the in vivo pharmacological activity of Centella asiatica acid derivatives. Some researchers have found that the fat solubility of the bioactive ingredients in Centella asiatica is low, and that they have poor ability to cross the intestinal membrane, which further reduces their oral bioavailability. In response to this problem, if Centella asiatica is prepared into a new formulation for clinical application, can the in vivo pharmacological activity of Centella asiatica be increased to achieve better clinical therapeutic effects? However, there has been little research on the pharmacological activity of Centella asiatica in vivo. The author believes that this can be a research direction for further study.

Centella asiatica has been recorded in the Shennong Bencao Jing (Shennong's Classic of Materia Medica) as early as the 5th century. It has been used in medicine for thousands of years and has been proven to be effective. However, only two proprietary Chinese medicines containing centella asiatica are included in the 2015 edition of the Pharmacopoeia of the People's Republic of China, indicating that centella asiatica still has great research value and broad application prospects in the clinic. In future research, we should use modern techniques and methods to study the in vivo activity of new Centella asiatica preparations, explore their bioavailability, and conduct more in-depth and extensive preclinical research, in order to play a positive role in the development of new Centella asiatica drugs.

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