A Comprehensive Review on Amorphophallus Paeoniifolius Tuber Extract: Phytochemistry and Pharmacological Potential

Elephant foot yam, or Amorphophallus paeoniifolius, is a tropical tuber crop of the Araceae family with great promise. Due to its potential productivity and culinary qualities, it is a significant tuber crop in tropical and sub-tropical nations¹. Southeast Asian nations including Malaysia, Indonesia, Philippines and India grow and eat elephant foot yams extensively².  Its great production potential, market acceptability and profitable economic returns (50–80 t/ha) have earned it the status of a cash crop in India. It is widely used as a vegetable in many Indian cuisines and is a good source of both protein and carbohydrates¹. In the human diet, elephant foot yam (EFY) corms can be used as a source of dietary fiber, resistant starch, protein, minerals, and vitamins³. It is grown in Andhra Pradesh, West Bengal, Gujarat, Kerala, Tamil Nadu, Karnataka, Maharashtra, Uttar Pradesh, and Jharkhand¹.

The crop is often grown as an intercrop with turmeric and under coconut or banana plantations. Recently, farmers in Bihar and Uttar Pradesh have also started growing it. Elephant foot yams demand well-aerated soils, they thrive in medium to light soils (sandy soils with a coarse texture) that contain sufficient levels of organic matter. While anaerobic waterlogging results in corm rot, the crop can withstand brief flooding⁴. Tubers are used as digestive, appetizer, stomachic, anthelmintic, liver tonic, aphrodisiac, emmenagogue, haemostatic, expectorant, carminative, anodyne, anti-inflammatory, anti-haemorrhoidal, and rejuvenating⁵.  Traditionally, they have been used to treat a variety of conditions, including arthralgia, elephantiasis, anorexia, flatulence, colic, constipation, helminthiasis, hepatopathy, spleenopathy, amenorrhea, dysmenorrhea, fatigue, anaemia, tumors⁵, asthma⁶, inflammations, haemorrhoids, haemorrhages, dyspepsia⁷. According to reports, the tuber possesses analgesic, antiprotease, central nervous system depressive⁵, anti-inflammatory⁸, cytotoxic⁹, antibacterial activity and antifungal activity¹⁰.

Amorphophallus species contains mainly more than 15 biological species Table 1

Table no. 1: Different species of Amorphophallus

VERNACULAR NAMES

Table no. 2: Vernacular names of Amorphophallus paeoniifolius

PLANT PROFILE:

• Kingdom: Plantae
• Phylum: Magnoliophyta
• Division: Angiosperms
• Class: Monocots
• Order: Alismatales
• Family: Araceae
• Genus: Amorphophallus
• Species: A. paeoniifolius
• Synonyms: A. campanulatus

MORPHOLOGICAL CHARACTERSTICS:

Leaf:

leaflets that are acuminate, elliptic, elliptic-oblong, elliptic-lanceolate or lanceolate, rounded, oval, ovate, obovate⁵.

Leaf-blade:                                                                       

leaf blade is located on the petiololus, or leaflets; fresh leaf blades are green throughout growth phases, but as they age, they turn yellow and eventually wilt with the petiole.  hairless; gloomy (laevis) surface; pinnate leaf bones (penninervis); flat-leaf edge (integer); thin, soft flesh (intervenum); tapering leaf tips (acuminatus); ellipticus/ovalis; widest point in the center; length reaches 31 cm; width 9 cm; number of leaf blades exceeds 816¹¹.

Petiole:

Green petiole with white spots¹², spherical, slightly rough on the surface, sticky or slimy, transparent, and irritating; the height up to 204 cm, canopy width of 294 cm, petiole tip diameter is 11 cm, and petiololus is 3¹¹.

Stem:

It is a dark brown, depressed-globose tuber with thick, rhizomatous off lets generated every season and noticeable⁵.

Tuber:

The tuber's exterior layer is dark brown-black in color, its flesh is reddish-white or yellowish-white, it is sticky or slimy, clear, and irritating. It is spherical and flat, with a groove in the middle where the petiole grows or appears, and it has no smell¹¹.

Flowers:

The inflorescence of flowers is short-peduncled; the peduncle is 3–20 cm long, 1–8 cm in diameter, and typically smoother and paler than the petiole. The spathas have a threaded spadix, maroon or purple in colour, and a carcass-like odour. They bloom for around a week^5,11.

Figure no: 1

PHYTOCHEMICAL CONSTITUENTS:

The phytochemical constituents of the plant contain alkaloid, flavonoid, steroid, tannin, carbohydrate, reducing sugar, amino acids, glycosides, saponins, phenols, glucomannans^13,14.

Table no. 3: Phytochemicals present in different parts of Amorphophallus paeoniifolius

Name of constituent	Structure
Quercetin
p-coumaric acid
Gallic acid
Resveratrol

PRELIMINARY PHOTOCHEMICAL SCREENING:

Qualitative chemical analysis was conducted on all extracts of Amorphophallus paeoniifolius to identify the presence of various phytoconstituents, as detailed below¹⁵.

Test for Alkaloids

Test for Flavonoids

Test for Glycosides

Test for Tannins and Phenolic compounds

A phytochemical screening of the methanol extract revealed the presence of flavonoids, alkaloids, carbohydrates, and steroids¹³.

The presence of steroids, lipids, and fixed oil is detected by phytochemical screening of petroleum ether extract⁵.

PHARMACOLOGICAL ACTIVITY:

Amorphophallus paeoniifolius plant species shows various activities as that in picture.

Figure no. 2

Analgesic activity:

Analgesics act on the central nervous system or peripheral pain mechanisms to selectively reduce pain without appreciably changing consciousness¹⁶. Yadu Nandan Dey et al., collected and authenticated the tuber of Amorphophallus paeoniifolius. The plant's tuber was dried in the shade, grind into a fine powder, and then extracted with methanol using a Soxhlet extractor. After weighing the isolated fraction, the yield percentage was determined to be 9.81%. Analgesic activity of Amorphophallus paeoniifolius tuber methanol extract and its fractions was evaluated using Male Swiss Albino mice (20-25 g).  The animals were kept in regular conditions with a 12-hour light and 12-hour dark cycle at 25°C, and they were fed with a standard diet. The tail flick method (using analgesiometer) and the acetic acid-induced writhing responses in mice were used to assess the analgesic activity. The Amorphophallus paeoniifolius methanol extract was given as a suspension in 5% v/v Tween 80 as the vehicle. Diclofenac sodium, a standard drug, was given as an injection in the form of a water solution. Male animals were split up into five groups, each consisting of six animals, to evaluate the analgesic activity of each approach. An intraperitoneal injection (up to 1ml under ethical guidelines) was given to each group.

• Group I: 10 mg/kg of 5% Tween 80 were given to the control animals.
• Group II: 250 mg/kg of methanol extract was given to the animals.
• Group III: 500 mg/kg of methanol extract was given to the animals.
• Group IV: Standard Diclofenac sodium (5 mg/kg) was administered to the animals.
• Group V: Standard Diclofenac sodium (10 mg/kg) was administered to the animals.

Using an Analgesiometer, the tail-flick method recorded the flicking reactions of each animal's tail one hour after the drug was administered to each group. Mice exhibited a writhing response to acetic acid, each mouse received intraperitoneal injections of 1% acetic acid (1 ml/100 g of animal body weight) one hour after the drug was administered. After five minutes, each group of mice's writhing responses were observed for ten minutes. Then the data were compared with the control groups.The obtained data was expressed as Mean ± SD. In these investigations, ANOVA and Dunnett's test were used to determine whether the differences between the control and treatment groups were significant. P-values less than 0.05 were deemed statistically significant¹³.

Table no.4: Analgesic activity of Amorphophallus paeoniifolius by Tail-Flick Method in Mice.

n=6 Values are Mean ± SD in each. Comparison was made between group I Vs II, III, IV, V and values *p>0.05, **p<0.01.

Figure no. 3: Dose response comparison by using Tail-Flick Method

n=6 *P<0.05 and **P<0.01 as compared to control.

Methanolic extract of Amorphophallus paeoniifolius tubers administered intraperitoneally at doses of 250 and 500 mg/kg produced a notable analgesic effect in the tail flick method. In the case of diclofenac, the percentage increase in analgesic activity is 65.3 (P<0.01), 82.7 (P<0.01), and n=6, respectively, whereas the percentage increase in analgesic activity is 58.10 (P<0.05), 78.90 (P<0.01), and (n=6).

Table no.5: Analgesic activity of Amorphophallus paeoniifolius by Acetic Acid Induced Writhing Response in mice

n=6, Values are Mean±SD. Comparison were made between group I Vs II, III, IV, V values *p<0.05, **p<0.01.      

Figure no. 4: Acetic Acid Induced Writhing Response Comparison

n=6 *P<0.05 and **P<0.01 as compared to control.

Amorphophallus paeoniifolius tuber methanolic extract (250, 500 mg/kg) administered intraperitoneally produced a notable analgesic effect in a dose-dependent manner in the acetic acid-induced writhing response method. The inhibitions percentage are 47.9 (P<0.05), 69.3 (P<0.01), and (n=60) respectively, whereas the percentage inhibitions for diclofenac are 70.78 (P<0.01), 81.86 (P<0.01), and 6 (n=6). Analgesic action peaked at 60 minutes.

The study indicates that the methanolic extract of the tubers of Amorphophallus paeoniifolius, which may have analgesic properties. Additionally, it was determined that the extract exhibited both cerebral and peripheral analgesic action.  The plant may include phytoconstituents that act on central opioid receptors (µ receptors) to reduce analgesia centrally or inhibit the cyclooxygenase enzyme to reduce analgesia peripherally. Diclofenac sodium, a standard drug, affects the cyclooxygenase pathway of prostaglandin production. Another possibility is that the analgesic action is caused by the benzodiazepine receptor agonistic activity, which depresses pain receptors¹³.

In vitro Antioxidant activity:

The Amorphophallus paeoniifolius tuber was collected and authenticated. The air-dried tuber powder was extracted with methanol and 70% of hydroalcohol solvents separately using soxhlet extractor. Afterwards, the solvent was evaporated using a rotary vacuum evaporator set below 50°C temperature e to produce a dark brownish 70% hydroalcoholic extract (AE) and reddish-brown methanol extract (ME). Since methanolic and 70% hydroalcoholic extracts contain tannins, phenols, flavonoids, triterpenoids, and coumarins, the current study aims to assess the antioxidant activity of these extracts. In order to determine the total phenolic content, working stock solutions of the extracts (ME & AE) were made to an appropriate concentration for analysis using distilled water. After making 50µl of extracts (ME: 25 mg/ml; AE: 65 mg/ml) to 3 ml with distilled water, 0.5 ml of Folin-Ciocaltean Phenol reagent was added, and the mixture was incubated for 3 minutes at room temperature. After adding 2 ml of a 20% w/v sodium carbonate solution, the mixture was incubated in a boiling water bath for one minute. The Shimadzu model UV 150-02 double beam spectrophotometer was used to detect absorption at 650 nm in relation to a reagent blank.

Table no. 6: Total phenolic content of extracts

Values are in mg equivalent to catechol per gram of extract; values are mean ± S.D.

1,1-diphenyl di-picrylhydrazil (DPPH) was used to assess the free radical scavenging activities of ME and AE. Antioxidants, also known as free radical scavengers, lowers the DPPH radicals, and the degree to which DPPH’s violet color is reduced is directly correlated with the free radical scavenging activity. BHA (Butylated hydroxyl Anisole) as a standard and extracts at several concentrations (10µg to 500µg) in 100 ml of methanol were taken, and 5µL of 0.1 mM DPPH in methanol was added. Shaken well and left to stand for 20 minutes at 27°C. The absorbance was then measured in a spectrophotometer using DPPH solution as a blank at 517 nm. A reaction mixture with a lower absorbance showed more free radical scavenging activity.

The scavenging activity is expressed as percentage of inhibition calculated by using equation:

% Antioxidant activity = {(control Abs-Sample Abs)/control Abs} x 100.

Table no.7: Free radical scavenging activity of ME, AE and BHA in DPPH method

Conc: Concentration; Values are mean ± standard deviation

Fig no. 5: Percentage inhibition of DPPH radicals by BHA and different doses of ME and AE.

Table 7 and Figure 5 summarized the DPPH scavenging activities exercised by ME & AE and BHA. In a dose-dependent manner, ME & AE showed 42.83% and 72.13% inhibition at 500 µg, while standard compound BHA showed 79.23% inhibition at 100 µg. Accordingly, in the current investigation, AE at higher doses showed better DPPH scavenging activity than ME.  

In this study indicates that both methanolic and 70 % hydroalcoholic extracts exhibits antioxidant properties which may be attributed to the antioxidant principles including flavonoids, phenols, tannins, coumarins etc. present in them. Since phenolics provide antioxidant activity, it stands to reason that drug or extract with a high TPC would have the highest antioxidant capacity¹⁷.

Antibacterial activity:

The antibacterial properties of A. paeoniifolius extracts have been tested against a variety of microorganisms, including Bacillus subtilis and Bacillus megaterium (Nayak A et al.,). The purpose of the study was to evaluate and contrast the antibacterial activity of A. paeoniifolius aqueous and ethanolic extracts against periodontal pathogens. Amorphophallus paeoniifolius tuber was collected and authenticated. The plant tuber was dried in shade and powdered. Using the Soxhlet extraction method, A. paeoniifolius aqueous and ethanolic extracts were prepared. Antibacterial activity was tested against Standard strains of Porphyromonas gingivalis (ATCC 33227), Prevotella intermedia (JCM 11150), and Fusobacterium nucleatum (ATCC 25886). To determine the minimum inhibitory concentrations of tetracycline hydrochloride and A. paeoniifolius extracts, the serial broth dilution method was used. The stock solutions is prepared by mixing 200 mg of prepared aqueous and ethanolic extracts in 1 ml of distilled water. To investigate the aqueous extract of A. paeoniifolius's minimal inhibitory concentrations (MIC), a set of 12 sterile vials were serially labeled 1 to 12. A 400 ml dilution of the stock solution, or aqueous extract, of A. paeoniifolius was added in tube number 1. In each tube (2 to 12) 200 ml of thioglycolate broth was added. A 10 ml of bacteria suspension was added to tubes (1 to 10) and tube 12. No organism was added to tube 11. The 200 ml of A. paeoniifolius extract from tube number 1 were added to tube number 2 and thoroughly mixed. Now, this mixed solution was added to tube number 3 and well mixed. This was carried on until tube number 11 and lastly 200 ml of mixture was removed from tube number 11. Tube number 11 was known as a broth control (to ascertain sterility check) because it contained broth and extract but no organism. Tube number 12 was regarded as an organism control (to check organism growth) because it contained broth and organism but no extract. A. paeoniifolius aqueous extract concentrations100, 50, 25, 12.5, 6.25, 3.1, 1.6, 0.8, 0.4, and 0.2 mg/ml, obtained.  After that, the tubes were incubated at 37°C for 48 hours. A visual examination was performed to ascertain the MIC values following the incubation. The same method was carried out for ethanolic extract of A. paeoniifolius and tetracycline hydrochloride. Tetracycline hydrochloride acts as a standard control group in the present investigation. Growth of the bacteria was shown by turbidity in the MIC tube, suggesting that the bacteria were resistant to the A. paeoniifolius aqueous extract. After a 24-hour incubation period, the tubes that were determined to be MIC-sensitive were plated, and the colony count was recorded.  MBC was used to determine whether the extracts and tetracycline hydrochloride had a bacteriostatic or bactericidal effect on the chosen organisms.  It was thought to have bactericidal effects if there was no growth.  It was determined that it was bacteriostatic if there was growth.

All organisms shown sensitivity to an aqueous extract of A. paeoniifolius at 0.4 mg/ml of .  P. gingivalis and F. nucleatum were shown to be sensitive to the ethanolic extract of A. paeoniifolius at 0.2 mg/ml, whereas P. intermedia was found to be sensitive at 0.4 mg/ml.  It was discovered that F. nucleatum was sensitive to tetracycline hydrochloride at 0.4 mg/ml, while P. gingivalis and P. intermedia were sensitive at 0.2 mg/ml. As shown in table no. 8 and figure no. 6.

Table no.8: Minimum bactericidal concentrations of Amorphophallus paeoniifolius extracts (aqueous and ethanolic) and tetracycline hydrochloride in mg/ml against Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum.

Figure no.6: minimum bactericidal concentration of Amorphophallus paeoniifolius extracts (aqueous and ethanolic) at 0.2 and 0.4 mg/ml concentration against Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), and Fusobacterium nucleatum (Fn). AEAP – Aqueous extract of Amorphophallus paeoniifolius; EEAP – Ethanolic extract of Amorphophallus paeoniifolius.

Anthelmintic activity:

Anthelmintics are medications that work by either eliminating (destroying) parasitic worms (vermicide) or driving them out of the body (vermifuge). Yadu Nandan Dey et al., collected the plant tuber and authenticated. The plant tuber was ground into fine powder in a lab mill and extracted with methanol using Soxhlet apparatus. The presence of steroids, flavonoids, alkaloids, and carbohydrates was shown by phytochemical screening of the methanolic extract. Amorphophallus paeoniifolius tuber methanolic extracts were evaluated for their anthelmintic properties against adult Indian earthworms, Tubifex tubifex and Pheretima posthuma because of their morphological and physiological similarities to the human intestinal roundworm parasite. In 20 ml formulations with three different concentrations, a methanolic extract of tuber (25, 50, and 100 mg/ml in double-distilled water) were made, put in various petridishes and then six earthworms of the same kind were added to each solution. Tubifex worm lumps were also added to the test solutions. All the test and standard drug solutions were freshly prepared before the experiments began. Paralysis time was recorded when the worms showed no movement at all, except when subjected to vigorous shaking. After it was determined that the worms did not move when shaken violently or when submerged in warm water at 50°C, the time it took for them to die was noted.  Distilled water served as the control and piperazine citrate (10 mg/ml) as the reference standard. Three sets of experiments were conducted in order to determine statistical significance.

Table no.9: Anthelmintic activity of methanolic extract of Amorphophallus Paeoniifolius

It was discovered that the average paralyzing time for Tubifex at doses of 25, 50, and 100 mg/ml was 94.66, 52.00, and 12.33 minutes, respectively.  Meanwhile, in 22.66 minutes, a dose of 10 mg/ml of piperazine citrate paralyzes the above helminth and a dosage of 10 mg/ml of piperazine citrate kills the above-mentioned helminth in 45.33 minutes¹⁹.

Anticancer activity:

Using A549 (Lung Cancer) cells, an in vitro anticancer investigation was conducted on the crude sample extracted from the tuber of Amorphophallus paeoniifolius. The cell line was planted into a 96-well tissue culture plate and kept in a humidified incubator with 5% CO2 at 37ºC. Compound stock preparation involved weighing 1 mg of the material and dissolving it in 1 mL of DMEM using a cyclomixer. To ensure sterility, the sample solution was filtered using a 0.22 µm millipore syringe filter. The growth medium was removed after 24 hours and the newly made compounds in 5% DMEM were serially diluted five times using a two-fold dilution (100 µg, 50 µg, 25 µg, 12.5 µg, and 6.25 µg in 500 µl of 5% DMEM). Each concentration was then added in triplicate to the corresponding wells and the wells were then incubated at 37 °C in a humidified 5% CO2 incubator. The Anticancer activity assay by MTT method procedure was completely described by M S Lata et al.,²⁰. The growth inhibition percentage was determined using formula:

% of viability = (Mean Optical Density of Samples/Mean Optical Density of control group) x100

Table no.10: Percentage viability of lung cancer cell lines with increase in sample Concentration.