Ribosome inactivating proteins (RIPs), are plant proteins with N Glycosidase activity. Annona squamosa is traditionally used in medicine but RIP presence is less explored. The present study aimed to isolate and structurally analyse RIP- like protein from the seeds of Annona squamosa (ARIP) and investigate its in vitro pharmacological bioactivities.

ARIP was isolated with step wise purification procedures – Cation exchange and Gel filtration column chromatography, Ultrafiltration and SDS PAGE were adopted to purify and separate ARIP. The protein bands were eluted and tested for the presence of ARIP by N Glycosidase activity. The trypsin digested peptides were characterized by LC-MS/ESI-MS and searched for match using MASCOT search engine. Pairwise sequence alignment of peptide sequences were attempted to predict the similarity with known RIP in Magnoliophyta. In vitro antimicrobial, antimutagenic and cytotoxic activity of the the purified fraction of the ARIP was also studied.

Two proteins with band sizes 21 kDa and 28 KDa confirmed to have ARIP like activity. The LC-MS/ESI-MS mass spectra between 9 and 17 min yielded 19 peaks for 21 kDa protein and 18 peaks for 18 peaks for 28 kDa protein. Although database search revealed very low similarity with known RIPs, the closest similarity observed was with Populus trichocarpa which is known to have Type 2 Ricin B chain RIP. On sequence alignment with RIPs, the peptides revealed similarity with RIPs of type II category. Phamacological investigation also suggested strong in vitro antimicrobial, antimutagenic and cytotoxic activity, proving to be a promising candidate as drugs in future.

Our study have observed that ARIP was novel protein with unique sequence but lectin binding properties. They have shown remarkable antimicrobial, antimutagenic and cytotoxic activity. Crystallization of the protein followed by X-ray characterization could help to study the structure of the protein, active sites which would in turn give clear focus on the usage of this RIP in treatment of various diseases.

核糖体失活蛋白（RIP）是具有N糖苷酶活性的植物蛋白。Annona squamosa在医学上传统上使用，但对RIP存在的探索较少。本研究旨在从鳞状Annona squamosa（ARIP）的种子中分离和结构分析RIP样蛋白，并研究其体外药理生物活性。

通过逐步纯化程序分离ARIP-通过阳离子交换和凝胶过滤柱色谱，超滤和SDS PAGE纯化和分离ARIP。洗脱蛋白条带并通过N糖苷酶活性测试ARIP的存在。胰蛋白酶消化的肽通过LC-MS / ESI-MS进行表征，并使用MASCOT搜索引擎进行匹配搜索。尝试对肽序列进行成对序列比对，以预测与Magnoliophyta中已知RIP的相似性。还研究了ARIP纯化部分的体外抗菌，抗诱变和细胞毒性活性。

两种大小分别为21 kDa和28 KDa的蛋白质具有类似ARIP的活性。9至17分钟之间的LC-MS / ESI-MS质谱图得出21 kDa蛋白的19个峰和28 kDa蛋白的18个峰的18个峰。尽管数据库搜索显示与已知RIP的相似性非常低，但是观察到的最接近的是与已知具有2型蓖麻毒素B链RIP的毛果杨。在与RIP进行序列比对时，这些肽与II型RIP具有相似性。药理学研究还表明，其强大的体外抗菌，抗诱变和细胞毒性活性，被证明是未来有望成为药物的候选药物。

我们的研究已经观察到ARIP是具有独特序列但具有凝集素结合特性的新型蛋白质。它们显示出显着的抗微生物，抗诱变和细胞毒性活性。蛋白质的结晶，然后进行X射线表征，可以帮助研究蛋白质的结构，活性位点，进而明确关注该RIP在治疗各种疾病中的用途。