A mini review on the analytical method and its validation for Psidium guajava

: The well-known tropical tree Psidium guajava , also known as the guava, is widely farmed for its fruit. This article consists of literature about various analytical techniques like TLC, UV spectrophotometry, HPLC, and HPTLC that could be used to analyze the Guava plant. The HPLC analysis of Quercetin isolated from the leaves of the guava plant uses a 4 x 125 mm Hypersil ODS column with a mobile phase of 0.5 percent ortho-phosphoric acid in water and methanol at a flow rate of 1ml/min. The column wash times are 20 min and 18 min, respectively. 25 0 C is the temperature in the column. Silica gel 60F 254 pre-coated plates (10x10 cm) were used with toluene: acetone: formic acid (38:10:5) as the solvent system for HPTLC analysis of quercetin obtained from the tender leaves of guava. The λ max was observed in the U.V. spectrum of the isolated compound (quercetin) on three standard wavelengths: 212 nm, 256 nm, and 372 nm. An effort can be made to produce a new analytical method that will be cost-effective and time-saving.


Introduction
Natural products from plants, animals, and minerals have been the basis of the treatment of human disease. The World Health Organization recommends and encourages the use of traditional herbs/remedies because a huge amount of raw material is easily available (1). Psidium guajava has a long history of being used medicinally. Common names for the plant include "Guava" in English, "Guayabo" in Spanish, "Goyave" and "Goyavier" in French, "Guyabaorgoeajab" in Dutch, "Goiaba" and "Goaibeira" in Portuguese, and "Jambubatu" in Malaya. P. guajava is a 6-to 25-foot-tall evergreen shrub or tree with leaves, flowers, fruit, seeds, bark, and other parts (2). The images of guava fruit, guava leaves, and guava tree is given in Figure 1. Guava leaves are abundant in flavonoids, especially quercetin. P. guajava leaves have recently yielded five ingredients, including a new pentacyclic triterpenoid called guajanoic acid and four previously identified substances called beta-sitosterol, uvaol, oleanolic acid, and ursolic acid. There are significant levels of tannins (11-27 %) in the guava tree's bark.
Leucocyanidin, luectic acid, ellagic acid, and amritoside have been identified from the stem bark (5). The alcoholic root bark of P. guajava contains flavonoids, tannins, alkaloids, and saponins (6). Guava fruit was also reported to contain carotenoids (7). A total of 87.06 % of guava seed oil was found to be primarily composed of unsaturated fatty acids, namely oleic acid and linoleic acid. In the guava seed oil, monounsaturated fatty acids makeup about 10% of the total unsaturated fatty acids, and polyunsaturated fatty acids made up 77% present in the guava seed oil (8,9).
P. guajava exhibits a wide range of pharmacological activities. The antioxidant activity of guava prevents lipid peroxidation (10). The water extract from the plant decreases the frequency of cough (11). The water extract of the plant shows anti-diabetic activity which possesses a hypoglycaemic effect (12). The aqueous and methanolic extracts of P. guajava bark were reported to show antibacterial properties (13). The aqueous leaf extracts from P. guajava exhibit good hepatoprotective action (14). Rats and mice were significantly protected against castor oil-induced diarrhoea by P. guajava leaf aqueous extract (15). The active flavonoid guaijaverin has shown excellent potential as an antiplaque agent (16). Direct-acting mutagens' mutagenicity was successfully inactivated by the P. guajava aqueous extract (17). P. guajava leaf extract reduces myocardial inotropism in Guinea pigs (18). The aglycone, quercetin showed spasmolytic activity (19). An aqueous extract of the budding leaves of guava showed anti-cancer activity (20). P. guajava leaf aqueous extract has analgesic and anti-inflammatory effects (21). Acne is treated with leaf extracts from P. guajava (22). The leaves of P.

UV of Quercetin
A total of 0.1g of quercetin (1000 µg/ml) was dissolved in 100 ml of ethanol. After diluting the 0.1ml of stock solution with 100ml distilled water (10 µg/ml), the  max value was seen in Table 2.

High Performance Thin Layer Chromatographic Method for Quantitative Determination of Quercetin in Tender Leaves of P. guajava
To quantify quercetin in P. guajava, the following parameters can be used:  Sample preparation: For the study, each region's crystal guava was cut into five pieces. After cleaning, the fruit's flesh was processed into a slurry with a juicer. Once the fruit has been processed, 10 grams of the sample was carefully weighed and it was placed in a 10 mL flask with the appropriate amount of aquabidest.

Validation and Vitamin C Testing in Crystal
It was centrifuged for 5 minutes at a speed of 5000 rpm. After centrifugation, the supernatant was filtered through a filter paper (Whatman no. 42) (31).
Determination of vitamin C: An extract of up to 100 μL of clear samples was taken into a 10 mL flask, and the appropriate amount of aquabidest was added. The samples were filtered with a 0.22 μm millipore paper. Then, 20 mL of the samples was injected into the HPLC system (31,33).

System Suitability Test
Based on various metrics, the coefficient of variation (CV) for the retention time was 1.94% ± 2%, and the CV for the area under the curve (AUC) was 1.523% ± 5%. The average values for N and the tailing factor are 5.7% and 15%, respectively. These results indicate the strong system compatibility of the HPLC method (31,34).

Linearity
The level of linearity between vitamin C levels and the AUC area was determined by the correlation

Precision
CV values of 1.37%, 2.83%, and 0.93% were observed at concentrations of 1 mg/mL, 5 mg/mL, and 9 mg/mL, respectively. Since the CV result was below 5%, it confirms the accuracy of the procedure. These findings demonstrate that this technique meets the precision criteria (31).

Quantitative Analysis
This approach was chosen for its excellent selectivity, sensitivity, and rapid processing.   (37). Guava leaf extract was used on jelly sweets to prevent S. mutans from growing in sugar syrup with high moisture content (38). Herbal scrub of guava was used to treat wrinkles, redness, and acne on the skin (39). Guava leaf extracts hard candies are used to inhibit upper respiratory tract infection (40). Guava leaf extract herbal gel showed antibacterial and antifungal activity (41).

Pharmaceutics
A face wash gel was prepared with P. guajava seed extract which possesses antioxidant and antibacterial properties (42). The toothpaste containing an Alcoholic Extract of P. guajava Leaf was formulated (43). Guava extract capsule was formulated on heart function in dengue fever and dengue haemorrhagic fever patients (44).

Conclusion
P. guajava is one of the most "widely utilized" herbal medicine. Almost every part of the plant possesses a potential therapeutic effect on a wide range of diseases. The main purpose of this review was to elaborate the analytical method development on P. guajava species with reference to the identification, separation, and quantification of the active components that are responsible for its therapeutic activity. Through this literature, we recommend the development of a more robust analytical method that will be more costeffective and time-saving.

Funding
Not applicable.

Acknowledgment
Not applicable.

Conflict of Interest
There authors declare no conflict of interest.